CN103014030A - RNAi (Ribonucleic acid interference) carrier of three genes of collard DWARF, SP and GA20ox, and building method and application thereof - Google Patents

Abstract

The invention discloses three gene segments (sequences are respectively shown in the 1st-936th nucleotide in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3) of collard DWARF, SP and GA20ox, and an RNAi (ribonucleic acid interference) carrier of three genes of collard DWARF, SP and GA20ox built on the basis of the segments. The RNAi carrier is transformed into collard through agrobacterium; and expression of the three genes of the collard DWARF, SP and GA20ox in a transgenic positive plant are obviously reduced in comparison with that of a non-transgenic plant, and the plant type is miniaturization phenotype. Thus, new miniature collard product is obtained by total silence of the three genes of the DWARF, SP and GA20ox. The RNAi carrier has good market prospect and economical value, and meanwhile, consultation and reference are provided for miniaturization breeding of other plants.

Description

Kale DWARF, SP and GA20ox three gene RNAi carriers and construction process and application

Technical field

The invention belongs to gene engineering technology field, relate to plant gene fragment, RNAi carrier, RNAi Vector construction method, and the application of RNAi carrier in plant breeding.

Background technology

Kale is the biennial herb plant, is the horticultural variety of edible wild cabbage (Caulis et Folium Brassicae capitatae, cabbage), and its leaf look enriches changeable, leafly also is not quite similar, and therefore whole plant shape such as tree peony are called again " leaf tree peony " visually, are the good merchantable brands of potted plant sight leaf.

Plant type is the important economical character of plant.Microminiaturized importance as the ornamental plant plant type has demonstrated good market outlook.For example, a special kinds---the miniature Chinese rose in the modern Chinese rose is because of the short and small compactness of its plant type, blade, flower are small and exquisite lovely, do not take up space, the advantage such as the putting position selectivity is larger, be loved by the people, become one of pot flowers most popular on the International Flower market at present.Therefore, cultivate the kale kind of plant type microminiaturization, estimate in the market in future, also can obtain good achievement.

Although traditional breeding method has been made huge contribution in the process that improves plant trait in the past, traditional breeding method is wasted time and energy.In recent years, develop day by day perfect with transgenic technology rapidly along with engineered, genetic engineering breeding has become an important branch of field of plant breeding.Bibliographical information, the plant type of tomato variety Micro-Tom is microminiaturized, and whole plant is compared whole scaled down with common tomato variety, and its plant type performance is mainly by two recessive mutation genes DwarfWith Self-pruningDetermine, wherein DWARFKey enzyme BR-6-oxydase in genes encoding brassinolide (important hormone of the regulating growth of plants) route of synthesis, SELF-PRUNINGThe developmental potentiality of gene major control shoot apical meristem transforms relevant with inflorescence.Document reports that also Plant hormones regulators,gibberellins also is the hormone that plant stem has been extended important regulating and controlling effect, and GA20oxIt is the key enzyme of its route of synthesis.

Summary of the invention

In view of this, one of purpose of the present invention is to clone kale DWARF, SPWith GA20oxThree gene fragments, two of purpose is to provide a kind of kale DWARF, SPWith GA20oxThree gene RNAi carriers, three of purpose is to provide described kale DWARF, SPWith GA20oxThree gene RNAi Vector construction methods, four of purpose are to provide and contain described kale DWARF, SPWith GA20oxThe microbial transformant of three gene RNAi carriers, five of purpose is to provide described kale DWARF, SPWith GA20oxThe application of three gene RNAi carriers in the breeding of kale plant, six of purpose are to provide and utilize described kale DWARF, SPWith GA20oxThe method that three gene RNAi carriers carry out the breeding of kale plant.

For achieving the above object, the invention provides following technical scheme:

1. kale DWARFGene fragment, nucleotide sequence is shown among the SEQ ID No.1 the 1st to the 936th.

2. kale SPGene fragment, nucleotide sequence is shown in SEQ ID No.2.

3. kale GA20oxGene fragment, nucleotide sequence is shown in SEQ ID No.3.

4. kale DWARF, SPWith GA20oxThree gene RNAi carriers, it contains a hpRNA expression cassette, described hpRNA expression cassette comprise the promotor that is linked in sequence, DWARF/ SP/ GA20oxThe forward fragment of three gene splicing fragments, DWARF/ SP/ GA20oxReverse fragment and the terminator of three gene splicing fragments; Described DWARF/ SP/ GA20oxThree gene splicing fragments comprise DWARFGene fragment or its complementary fragment, SPGene fragment or its complementary fragment and GA20oxGene fragment or its complementary fragment, described DWARFThe nucleotide sequence of gene fragment shown among the SEQ ID No.1 the 1st to the 936th, SPThe nucleotide sequence of gene fragment shown in SEQ ID No.2, GA20oxThe nucleotide sequence of gene fragment is shown in SEQ ID No.3.

Further, described hpRNA expression cassette comprise the CaMV35S promotor that is linked in sequence, DWARF/ SP/ GA20oxThe forward fragment of three gene splicing fragments, PDK intron, DWARF/ SP/ GA20oxThe reverse fragment of three gene splicing fragments and Ocs terminator; Described DWARF/ SP/ GA20oxThree gene splicing fragments comprise and being linked in sequence DWARFThe forward fragment of gene fragment, SPThe forward fragment of gene fragment and GA20oxThe reverse complemental fragment of gene fragment.

Further, described kale DWARF, SPWith GA20oxThree gene RNAi carriers are take the pBIN19 carrier as skeleton, and described hpRNA expression cassette inserts in the multiple clone site of pBIN19 carrier, and is described DWARF/ SP/ GA20oxThe nucleotide sequence of three gene splicing fragments is shown in SEQ ID No.4.

5. kale DWARF, SPWith GA20oxThree gene RNAi Vector construction methods may further comprise the steps:

A. kale DWARF, SPWith GA20oxThe clone of three gene fragments: take the total RNA of kale spire as template, reverse transcription obtains cDNA, again take gained cDNA as template, respectively take BoDF-F and BoDF-R, BoSP-F and BoSP-R, BoGA20ox-F and BoGA20ox-R as primer, the pcr amplification nucleotide sequence is shown in SEQ ID No.1 DWARFGene fragment, nucleotide sequence are shown in SEQ ID No.2 SPGene fragment and nucleotide sequence are shown in the SEQ ID No.3 GA20oxGene fragment; The nucleotide sequence of described primer BoDF-F and BoDF-R is respectively shown in SEQ ID No.5 and SEQ ID No.6, shown in SEQ ID No.7 and SEQ ID No.8, the nucleotide sequence of BoGA20ox-F and BoGA20ox-R is respectively shown in SEQ ID No.9 and SEQ ID No.10 respectively for the nucleotide sequence of BoSP-F and BoSP-R; Then, with the pcr amplification gained DWARFGene fragment and SPGene fragment is connected with the pMD18-T carrier respectively, obtains recombinant plasmid DWARF::pMD18-T and SP::pMD18-T, in addition with the pcr amplification gained GA20oxGene fragment is connected with pGEM-T Easy carrier, obtains recombinant plasmid GA20ox::pGEM-T Easy;

B. kale DWARF, SPWith GA20oxThe splicing of three gene fragments: recombinant plasmid DWARF::pMD18-T is used XhoI and EcoThe RI double digestion reclaims purification of Recombinant plasmid DWARF::pMD18-T linear fragment, in addition recombinant plasmid SP::pMD18-T is used SalI and EcoThe RI double digestion reclaims purifying SPGene fragment, again with the recombinant plasmid DWARF::pMD18-T linear fragment of purifying with SPGene fragment connects, and obtains recombinant plasmid SP/DF::pMD18-T; Then, recombinant plasmid SP/DF::pMD18-T is used XbaI and EcoThe RI double digestion reclaims purification of Recombinant plasmid SP/DF::pMD18-T linear fragment, in addition recombinant plasmid GA20ox::pGEM-T Easy is used SpeI and EcoThe RI double digestion reclaims purifying GA20ox gene fragment, and the recombinant plasmid SP/DF::pMD18-T linear fragment with purifying is connected with the GA20ox gene fragment again, obtains recombinant plasmid SP/DF/GA20ox::pMD18-T;

C. kale DWARF, SPWith GA20oxThree gene RNAi Vector constructions: take recombinant plasmid SP/DF/GA20ox::pMD18-T as template, take DWARF (F+EX) and GA20ox (F+KB) as primer, pcr amplification both sides band restriction enzyme site DWARF/ SP/ GA20oxThree gene splicing fragments; The nucleotide sequence of described primer DWARF (F+EX) and GA20ox (F+KB) is respectively shown in SEQ ID No.11 and SEQ ID No.12; The PCR product of purifying is used XbaI and BamThe HI double digestion reclaims purifying DWARF/ SP/ GA20oxThree gene splicing fragments, the pHANNIBAL carrier with the same double digestion of warp is connected again, obtains recombinant plasmid SP/DF/GA20ox::pHANNIBAL; In addition the PCR product of purifying is used KpnI and EcoThe RI double digestion reclaims purifying DWARF/ SP/ GA20oxThree gene splicing fragments, the recombinant plasmid SP/DF/GA20ox::pHANNIBAL with the same double digestion of warp is connected again, obtains to contain recombinant plasmid (SP/DF/GA20ox) i::pHANNIBAL of hpRNA expression cassette; Then, recombinant plasmid (SP/DF/GA20ox) i::pHANNIBAL is used SpeI and SacThe I double digestion reclaims purifying hpRNA expression cassette, in addition plant expression vector pBIN19 is used XbaI and SacThe I double digestion reclaims cmy vector pBIN19 linear fragment, and the carrier pBIN19 linear fragment with purifying is connected with the hpRNA expression cassette again, obtains recombinant plasmid (SP/DF/GA20ox) i::pBIN19, i.e. kale DWARF, SPWith GA20oxThree gene RNAi carriers.

6. contain kale DWARF, SPWith GA20oxThe microbial transformant of three gene RNAi carriers.

Further, described microorganism is Agrobacterium.

7. kale DWARF, SPWith GA20oxThe application of three gene RNAi carriers in the breeding of microminiaturized kale plant.

8. utilize kale DWARF, SPWith GA20oxThree gene RNAi carriers obtain the method for microminiaturized kale plant, may further comprise the steps:

A. the preparation of kale explant: after the kale seed disinfection, be seeded in that to contain massfraction be that 3% sucrose and massfraction are that 0.8% agar, pH are on 5.8 the 1/2MS solid medium, under 25 ± 2 ℃, photoperiod 16h/d, intensity of illumination 1000-2000lx condition, cultivate; 7-10 days Bands handle cotyledon of growth is downcut, it is that 3% sucrose, massfraction are 0.8% agar, 1mg/L 6-benzyl aminoadenine (6-BA) and 1mg/L 2 that access contains massfraction, 4-D, pH are in 5.8 the MS solid medium, preculture is 48 hours under 25 ± 2 ℃ of dark conditions, makes the kale explant;

B. the preparation of Agrobacterium engineering bacteria liquid: will contain kale DWARF, SPWith GA20oxThe Agrobacterium engineering strain of three gene RNAi carriers is inoculated in that to contain massfraction be 1.2% agar, 50mg/L Rifampin (Rif), 500mg/L Streptomycin sulphate (Str) and 50mg/L kantlex (Kan), pH is on 7.2 the YEB solid medium, activate 2-3 days to growing single bacterium colony under 28 ± 2 ℃ of dark conditions, picking list bacterium colony, with containing 50mg/L Rif, 500mg/L Str and 50mg/L Kan, pH is 7.2 YEB liquid nutrient medium 20mL, at 28 ± 2 ℃, enlarged culturing is 1.5 days under the 200rpm condition, again gained bacterium liquid is contained 50mg/L Rif for the 1:100 access by volume, 500mg/L Str and 50mg/L Kan, pH is in 7.2 the YEB liquid nutrient medium, at 28 ± 2 ℃, enlarged culturing is to OD under the 200rpm condition ₆₀₀=1.8-2.0,28 ± 2 ℃ of centrifugal supernatants of abandoning then, thalline be with fresh YEB liquid nutrient medium washing, is that 3% sucrose, pH are that 5.8 MS salts solution 100mL is resuspended with containing massfraction again, makes Agrobacterium engineering bacteria liquid;

C. Agrobacterium infect conversion: after the kale explant that step a is made is put and is contaminated 15 minutes in the Agrobacterium engineering bacteria liquid that step b makes, it is 3% sucrose that access contains massfraction, massfraction is 0.8% agar, 1mg/L 6-BA and 1mg/L 2,4-D, pH is in 5.8 the MS solid medium, cultivated altogether 48 hours under 25 ± 2 ℃ of dark conditions, change again that to contain massfraction be 3% sucrose over to, massfraction is 0.8% agar, 4mg/L 6-BA, 2mg/L zeatin (ZT), the 5mg/L Silver Nitrate, 500mg/L Pyocianil (Carb) and 20mg/L Kan, pH is in 5.8 the MS solid medium, at 25 ± 2 ℃, photoperiod 16h/d, be cultured under the intensity of illumination 1000-2000lx condition and grow green callus and resistant buds; The resistant buds of long 2-3cm is downcut, change that to contain massfraction be that 3% sucrose, massfraction are that 0.8% agar, 0.2mg/L naphthylacetic acid (NAA), 250mg/L Carb and 5mg/L Kan, pH are in 5.8 the 1/2MS solid medium over to, under 25 ± 2 ℃, photoperiod 16h/d, intensity of illumination 1000-2000lx condition, be cultured to and take root, namely get transgenosis kale plant;

D. the acquisition of transgenic positive plant: detect kale by PCR DWARF, SPWith GA20oxThe expression of the reporter gene that contains in the three gene RNAi carriers in transgenosis kale plant, and qPCR detects DWARF, SPWith GA20oxThe expression of three genes in transgenosis kale plant filters out the transgenic positive plant from the transgenosis kale plant that step c obtains, namely obtain microminiaturized kale plant.

Further, the kale kind is red No. 1 of capital lotus among the described step a.

Further, described kale DWARF, SPWith GA20oxContain the NPTII reporter gene in the three gene RNAi carriers, described steps d detects expression and the qPCR of NPTII reporter gene in transgenosis kale plant by PCR and detects DWARF, SPWith GA20oxThe expression of three genes in transgenosis kale plant comes the screening transgenic positive plant; Described PCR detection method is to get respectively transgenosis kale plant and the non-transgenic kale plant that step c obtains, extract genomic dna, again take the gained genomic dna as template, NPTII-F and NPTII-R carry out PCR as primer, the nucleotide sequence of described primer NPTII-F and NPTII-R is respectively shown in SEQ ID No.13 and SEQ ID No.14; Described qPCR detection method is to get respectively transgenosis kale plant and the non-transgenic kale plant that step c obtains, extract total RNA, be cDNA with total RNA reverse transcription, again take gained cDNA as template, respectively with Auele Specific Primer qBoDWARF-F and qBoDWARF-R, qBoSP-F and qBoSP-R, qBoGA20ox-F and qBoGA20ox-R, confidential reference items primer qBoactin-F and qBoactin-R carry out qPCR, the nucleotide sequence of described primer qBoDWARF-F and qBoDWARF-R is respectively shown in SEQ ID No.15 and SEQ ID No.16, the nucleotide sequence of qBoSP-F and qBoSP-R is respectively shown in SEQ ID No.17 and SEQ ID No.18, shown in SEQ ID No.19 and SEQ ID No.20, the nucleotide sequence of qBoactin-F and qBoactin-R is respectively shown in SEQ ID No.21 and SEQ ID No.22 respectively for the nucleotide sequence of qBoGA20ox-F and qBoGA20ox-R.

The substratum of 1/2MS described in the present invention is reduced to the MS substratum of original content 1/2 for other concentration of component except sucrose; Described MS salts solution is not for containing the MS liquid nutrient medium of organic components.

Beneficial effect of the present invention is:

(1) first passage genetic engineering technique of the present invention is developed the new variety of plant of plant type microminiaturization, transform by plant gene, but motivated, efficient and genetic stability ground has changed the plant plant type, avoided wasting time and energy, by spraying corresponding plant hormone, changing the traditional breeding way that plant farming time and cuttage mode obtain dwarf plant plant type that can not genetic stability, for positive exploration has been made in the breed improvement of ornamental plant.

(2) the present invention utilizes the spore homology, and the clone obtains kale DWARF, SPWith GA20oxThree gene fragments have made up kale based on above-mentioned three gene fragments DWARF, SPWith GA20oxThe coprecipitated silent RNAi carrier of three genes, and change this RNAi carrier over to kale, obtained the transgenic positive plant.Analytical results shows, in the transgenic positive plant DWARF, SPWith GA20oxTrigenic expression all significantly reduces than the non-transgenic plant, and its strain is microminiaturized phenotype, thereby passes through DWARF, SPWith GA20oxThe coprecipitated silent microminiaturized kale new variety that obtained of three genes.Kale provided by the invention DWARF, SPWith GA20oxThree gene fragments, kale DWARF, SPWith GA20oxThree gene RNAi carrier and construction processs thereof, the genetic transforming method of kale utilizes kale DWARF, SPWith GA20oxThe method that three gene RNAi carriers obtain microminiaturized kale plant all has good market outlook and economic worth, and the simultaneously microminiaturized breeding for other plant provides reference and reference.

Description of drawings

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing.

Fig. 1 is schematic flow sheet of the present invention.

Fig. 2 is kale DWARF, SPThe agarose gel electrophoresis analysis chart of two gene amplified fragments, wherein M is dna molecular amount standard (Maker), 1 is DWARFThe gene amplification fragment, 2 are SPThe gene amplification fragment.

Fig. 3 is kale GA20oxThe agarose gel electrophoresis analysis chart of gene amplification fragment, wherein M is Maker, 1 and 2 are GA20oxThe gene amplification fragment.

Fig. 4 is that recombinant plasmid DWARF::pMD18-T makes up synoptic diagram.

Fig. 5 is that recombinant plasmid SP::pMD18-T makes up synoptic diagram.

Fig. 6 is that recombinant plasmid GA20ox::pGEM-T Easy makes up synoptic diagram.

Fig. 7 is that recombinant plasmid SP/DF::pMD18-T makes up synoptic diagram.

Fig. 8 is that recombinant plasmid SP/DF/GA20ox::pMD18-T makes up synoptic diagram.

Fig. 9 is that recombinant plasmid SP/DF/GA20ox::pHANNIBAL makes up synoptic diagram.

Figure 10 is that recombinant plasmid (SP/DF/GA20ox) i::pHANNIBAL makes up synoptic diagram.

Figure 11 is that recombinant plasmid (SP/DF/GA20ox) i::pBIN19 makes up synoptic diagram.

Figure 12 is EcoRI and XbaThe agarose gel electrophoresis analysis chart of I double digestion recombinant plasmid (SP/DF/GA20ox) i::pBIN19, wherein contrast is for recombinant plasmid (SP/DF/GA20ox) i::pBIN19, and 1 and 2 cut product for enzyme, and M is Maker.

Figure 13 induces at resistance screening for the kale explant after cultivating altogether and grows green callus in the substratum of sprouting.

Figure 14 is the differentiation of resistant buds.

Figure 15 is transgenosis kale plant.

Figure 16 is that PCR detects the expression of NPTII reporter gene in transgenosis kale plant, and wherein M is Maker, and NT is without the template blank, the negative contrast of CK-, and the positive contrast of CK+, 1-6 is transgenosis kale plant.

Figure 17 is DWARF, SPWith GA20oxThe reticent effect identification of three genes in transgenosis kale positive plant, wherein 1 and 2 is transgenosis kale positive plant, WT is non-transgenic kale plant.

Figure 18 is the phenotype of transgenosis kale positive plant, and wherein T1 is transgenosis kale positive plant, and WT is non-transgenic kale plant.

Figure 19 is the process of growth phenotype of transgenosis kale positive plant, and wherein 1 and 2 is transgenosis kale positive plant, and WT is non-transgenic kale plant.

Embodiment

Below with reference to accompanying drawing the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in the preferred embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example, J. the work such as Pehanorm Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or the condition of advising according to manufacturer.

The kale kind of using in the preferred embodiment is red No. 1 of capital lotus.The pMD18-T carrier is TaKaRa company product, pGEM-T Easy carrier is Promega company product, and pHANNIBAL carrier, pBIN19 carrier, bacillus coli DH 5 alpha, intestinal bacteria " assistance " bacterium (helper) that contain the plasmid Prk2013 that moves about, agrobacterium tumefaciens lba4404 are preserved by this laboratory.RNA extracts test kit, RNA PCR Kit (AMV) Ver.3.0 test kit, DNA extraction test kit Universal Genomic DNA Extraction Kit Ver. 3.0, connects test kit DNA Ligation Kit Ver.2.0 that (T4 dna ligase/SolutionI), restriction enzyme, Taq archaeal dna polymerase, PrimeSTARHS archaeal dna polymerase are Dalian TaKaRa company product.It is TIANGEN company product that ultra-thin/plain agar sugar gel DNA reclaims test kit (centrifugal column type).All the other reagent are import or domestic analytical reagent.

Schematic flow sheet of the present invention as shown in Figure 1.

One, kale DWARF, SPWith GA20oxThe clone of three gene fragments

According to tomato DWARFGene order (GenBank accession number U54770), BLAST obtains in Arabidopis thaliana and rape (kale) database gene order of homology in TIGR database (http://compbio. dfci.harvard.edu/cgi-bin/tgi/Blast/index.cgi), at conservative region design Auele Specific Primer BoDF-F and the BoDF-R of these two species; According to tomato SPGene order (GenBank accession number U84140), BLAST obtains in Arabidopis thaliana and rape (kale) database gene order of homology in the TIGR database, at conservative region design Auele Specific Primer BoSP-F and the BoSP-R of these two species; According to Arabidopis thaliana GA20oxGene order (GenBank accession number X83379), BLAST obtains in rape (kale) database gene order of homology in the TIGR database, at conservative region design Auele Specific Primer BoGA20ox-F and the BoGA20ox-R of these two species; Primer sequence is as follows:

BoDF-F：5'-ATCGCTGCGGTTCACGG-3'(SEQ ID No.5)；

BoDF-R：5'-CTCCCATTTGTATCTCGTA-3'(SEQ ID No.6)；

BoSP-F：5'-CCCAAAGCAGAACTAAAGC-3'(SEQ ID No.7)；

BoSP-R：5'-TGATCTCATGTCACCCC-3'(SEQ ID No.8)；

BoGA20ox-F：5'-GGTCAATCACGGCATCAG-3'(SEQ ID No.9)；

BoGA20ox-R：5'-TCGGTGGCGTCACTACTC-3'(SEQ ID No.10)。

Take kale (red No. 1 of capital lotus) the total RNA of spire as template, according to RNA PCR Kit (AMV) Ver.3.0 test kit specification sheets, take the reverse transcription of oligodT primer as cDNA, again take gained cDNA as template, respectively take BoDF-F and BoDF-R, BoSP-F and BoSP-R, BoGA20ox-F and BoGA20ox-R as primer, the pcr amplification kale DWARF, SPWith GA20oxThree gene fragments.The PCR reaction system is: ddH ₂O 16.5 μ L, 10 * PCR Buffer, 2.5 μ L, MgCl ₂2.5 μ L, dNTP (10 μ M) 1.0 μ L, upstream and downstream primer each 0.5 μ L, cDNA template 1.0 μ L, Taq archaeal dna polymerase 0.5 μ L, totally 25 μ L.The PCR response procedures is: 94 ℃ of denaturations 5 minutes; Then 94 ℃ of sex change are 30 seconds, 56 ℃ of annealing 30 seconds, and 72 ℃ are extended 1 minute (extended 30 seconds as 72 ℃ during as primer take BoSP-F and BoSP-R, extended 50 seconds as 72 ℃ during as primer take BoGA20ox-F and BoGA20ox-R), totally 35 circulations; Last 72 ℃ were extended 10 minutes eventually.

The PCR product is identified after (Fig. 2-3) through agarose gel electrophoresis, adopts ultra-thin/plain agar sugar gel DNA to reclaim test kit and reclaim purifying.Kale with purifying DWARFGene fragment and SPGene fragment is connected with the pMD18-T carrier respectively, obtains recombinant plasmid DWARF::pMD18-T and SP::pMD18-T (Fig. 4-5); Kale with purifying GA20oxGene fragment is connected with pGEM-T Easy carrier, obtains recombinant plasmid GA20ox::pGEM-T Easy (Fig. 6).Entrust the English Weihe River prompt base (Shanghai) trade Co., Ltd to check order above-mentioned three kinds of recombinant plasmids, the result shows, obtains respectively the kale of 974bp DWARFThe kale of gene fragment (SEQ ID No.1), 336bp SPThe kale of gene fragment (SEQ ID No.2) and 710bp GA20oxGene fragment (SEQ ID No.3).

Two, Kale DWARF, SPWith GA20oxThe splicing of three gene fragments

Recombinant plasmid DWARF::pMD18-T is used XhoI and EcoThe RI double digestion reclaims purification of Recombinant plasmid DWARF::pMD18-T linear fragment; In addition recombinant plasmid SP::pMD18-T is used SalI and EcoThe RI double digestion reclaims purifying SPGene fragment; Again with the recombinant plasmid DWARF::pMD18-T linear fragment of purifying with SPGene fragment connects under the effect of T4 dna ligase, obtains recombinant plasmid SP/DF::pMD18-T (Fig. 7).Then, recombinant plasmid SP/DF::pMD18-T is used XbaI and EcoThe RI double digestion reclaims purification of Recombinant plasmid SP/DF::pMD18-T linear fragment; In addition recombinant plasmid GA20ox::pGEM-T Easy is used SpeI and EcoThe RI double digestion reclaims purifying GA20ox gene fragment; Recombinant plasmid SP/DF::pMD18-T linear fragment with purifying is connected under the effect of T4 dna ligase with the GA20ox gene fragment again, obtains recombinant plasmid SP/DF/GA20ox::pMD18-T (Fig. 8).Entrust the English Weihe River prompt base (Shanghai) trade Co., Ltd to check order recombinant plasmid SP/DF/GA20ox::pMD18-T, the result shows, obtains 1997bp's DWARF/ SP/ GA20oxThree gene splicing fragments (SEQ ID No.4) are comprising the kale of the 936bp that is linked in sequence DWARFThe forward fragment of gene fragment (the 1st to the 936th Nucleotide among the SEQ ID No.1), the kale of 336bp SPThe forward fragment of gene fragment (SEQ ID No.2) and the kale of 710bp GA20oxThe reverse complemental fragment of gene fragment (SEQ ID No.3).

In the above-mentioned steps, the enzyme system of cutting is: ddH ₂O 23 μ L, recombinant plasmid 20 μ L, each 1 μ L of restriction enzyme, 10 * Buffer, 5 μ L, totally 50 μ L; Linked system is: T4 dna ligase (SolutionI) 5 μ L, target gene fragment 4.5 μ L, recombinant plasmid linear fragment 0.5 μ L, totally 10 μ L.

Three, kale DWARF, SPWith GA20oxThree gene RNAi Vector constructions

Multiple clone site and gained according to the pHANNIBAL carrier DWARF/ SP/ GA20oxThree gene splicing fragment sequences, design following primer:

DWARF (F+EX): 5'-CCG GAATTC TCTAGAATCGCTGCGGTTCACGG-3'(SEQ ID No.11), underscore partly is XbaI restriction enzyme site, black matrix partly are EcoThe RI restriction enzyme site;

GA20ox (F+KB): 5'-CGG GGTACC GGATCCGGTCAATCACGGCATCAG-3'(SEQ ID No. 12), underscore partly is BamHI restriction enzyme site, black matrix partly are KpnThe I restriction enzyme site.

Take recombinant plasmid SP/DF/GA20ox::pMD18-T as template, take DWARF (F+EX) and GA20ox (F+KB) as primer, pcr amplification both sides band restriction enzyme site DWARF/ SP/ GA20oxThree gene splicing fragments.The PCR system is: ddH ₂O 16 μ L, 10 * PrimeSTARBuffer (contain MgCl ₂) 2.5 μ L, dNTP (2.5 μ M) 4.0 μ L, upstream and downstream primer each 0.5 μ L, plasmid template 1.0 μ L, PrimeSTARHS archaeal dna polymerase 0.5 μ L, totally 25 μ L.The PCR loop parameter is: 98 ℃ of sex change 10 seconds, and 56 ℃ of annealing 15 seconds, 72 ℃ were extended totally 40 circulations 2 minutes; Last 72 ℃ were extended 10 minutes eventually.The PCR product adopts ultra-thin/plain agar sugar gel DNA to reclaim test kit and reclaim purifying after agarose gel electrophoresis is identified.

The PCR product of purifying is used XbaI and BamThe HI double digestion reclaims purifying DWARF/ SP/ GA20oxThree gene splicing fragments, the pHANNIBAL carrier with the same double digestion of warp is connected under the effect of T4 dna ligase again, obtains recombinant plasmid SP/DF/GA20ox::pHANNIBAL (Fig. 9).In addition the PCR product of purifying is used KpnI and EcoThe RI double digestion reclaims purifying DWARF/ SP/ GA20oxThree gene splicing fragments, the recombinant plasmid SP/DF/GA20ox::pHANNIBAL with the same double digestion of warp is connected under the effect of T4 dna ligase again, obtains recombinant plasmid (SP/DF/GA20ox) i::pHANNIBAL (Figure 10), is about to DWARF/ SP/ GA20oxThree gene splicing fragments are forward and reverse both sides of inserting PDK intron in the pHANNIBAL carrier respectively, to form hpRNA expression cassette " 35S pro-fDWARF/SP/GA20ox-pdk-rDWARF/SP/ GA20ox-Ocs ter ".

Then, recombinant plasmid (SP/DF/GA20ox) i::pHANNIBAL is used SpeI and SacThe I double digestion reclaims the above-mentioned hpRNA expression cassette of purifying, in addition plant expression vector pBIN19 is used XbaI and SacThe I double digestion, reclaim cmy vector pBIN19 linear fragment, carrier pBIN19 linear fragment with purifying is connected under the effect of T4 dna ligase with the hpRNA expression cassette again, obtains recombinant plasmid (SP/DF/GA20ox) i::pBIN19 (Figure 11), i.e. kale DWARF, SPWith GA20oxThree gene RNAi carriers.Recombinant plasmid (SP/DF/GA20ox) i::pBIN19 is transformed the bacillus coli DH 5 alpha competent cell, blue hickie and Kan screening positive clone carry out single bacterium colony PCR with the carrier primer and detect, and extract recombinant plasmid after the positive bacterium colony enlarged culturing, carry out PCR with the carrier primer and detect, and use EcoRI and XbaI carries out double digestion and detects.Enzyme is cut the result and is shown, occurs 12491bp, 4821bp and 1383bp totally 3 electrophoretic bands after recombinant plasmid (SP/DF/GA20ox) i::pBIN19 enzyme is cut, and is consistent with notional result (Figure 12).

Four, kale DWARF, SPWith GA20oxThree gene RNAi carriers transform Agrobacterium

Agrobacterium tumefaciens lba4404 is rule at the YEB solid medium that contains 1.2% (w/w) agar, 50mg/L Rif and 500mg/L Str, cultivate 2-2.5 days under 28 ± 2 ℃ of dark conditions to growing single bacterium colony; The bacillus coli DH 5 alpha that will contain respectively recombinant plasmid (SP/DF/GA20ox) i::pBIN19 is rule at the LB solid medium that contains 50mg/L Kan with the intestinal bacteria helper that contains the plasmid Prk2013 that moves about, and is inverted under 37 ± 2 ℃ of conditions and cultivates 14-16 hour to growing single bacterium colony; With agrobacterium tumefaciens lba4404 list bacterium colony, the single bacterium colony of intestinal bacteria helper and the bacillus coli DH 5 alpha list bacterium colony that contains recombinant plasmid (SP/DF/GA20ox) i::pBIN19 are overlapping successively, and to be evenly coated in YEB solid medium (pH7.2) mid-diameter that contains 1.2% (w/w) agar be in the circle of 1cm, be inverted under 28 ± 2 ℃ of dark conditions and cultivate altogether 24 hours to growing cenobium, an amount of with transfering loop picking cenobium, containing 1.2% (w/w) agar, 50mg/L Rif, line in the YEB solid medium (pH7.2) of 500mg/L Str and 50mg/L Kan, be inverted under 28 ± 2 ℃ of dark conditions and cultivate 2-2.5 days to growing single bacterium colony, picking 3-4 single bacterium colony, with containing 50mg/L Rif, the YEB liquid nutrient medium (pH7.2) of 500mg/L Str and 50mg/L Kan is at 28 ± 2 ℃, dark, under the 200rpm condition shaking table cultivate 1.5 days to bacterium liquid evenly and OD ₆₀₀=1.8-2.0 extracts recombinant plasmid, uses EcoRI and XbaI carries out double digestion and identifies that positive recombinant is (SP/DF/GA20ox) i::pBIN19 Agrobacterium engineering strain, and-80 ℃ frozen for subsequent use.

Five, agriculture bacillus mediated DWARF, SPWith GA20oxThree gene RNAi carriers transform kale

With kale (red No. 1 of capital lotus) seed with 70% (v/v) alcohol solution dipping 1 minute, aseptic water washing 3 times, use again 1% (v/v) chlorine bleach liquor repeatedly to wash 12-15 minute, aseptic water washing 8 times, be seeded on the 1/2MS solid medium (pH5.8) that contains 3% (w/w) sucrose and 0.8% (w/w) agar, under 25 ± 2 ℃, photoperiod 16h/d, intensity of illumination 1000-2000lx condition, cultivate; The Bands handle cotyledon that launched (not growing true leaf) in 7-10 days fully of will growing downcuts, access preculture substratum (contains 3% (w/w) sucrose, 0.8% (w/w) agar, 1mg/L 6-BA and 1mg/L 2, the MS solid medium of 4-D, pH5.8) in, preculture is 48 hours under 25 ± 2 ℃ of dark conditions, makes the kale explant.

(SP/DF/GA20ox) i::pBIN19 Agrobacterium engineering strain is inoculated in contains 1.2% (w/w) agar, 50mg/L Rif, on the YEB solid medium (pH7.2) of 500mg/L Str and 50mg/L Kan, activate 2-3 days to growing single bacterium colony under 28 ± 2 ℃ of dark conditions, picking list bacterium colony, with containing 50mg/L Rif, YEB liquid nutrient medium (pH7.2) 20mL of 500mg/L Str and 50mg/L Kan, at 28 ± 2 ℃, enlarged culturing is 1.5 days under the 200rpm condition, get again and cultivate bacterium liquid 1mL, add and contain 50mg/L Rif, YEB liquid nutrient medium (pH7.2) 100mL of 500mg/L Str and 50mg/L Kan is at 28 ± 2 ℃, under the 200rpm condition enlarged culturing 8-10 hour to OD ₆₀₀=1.8-2.0, centrifugal 10 minutes of 28 ℃, 4000rpm, abandon supernatant, thalline washs with fresh YEB liquid nutrient medium (pH7.2), resuspended with MS salts solution (pH5.8) 100mL that contains 3% (w/w) sucrose again, make (SP/DF/GA20ox) i::pBIN19 Agrobacterium engineering bacteria liquid.

Aforementioned kale explant put in (SP/DF/GA20ox) i::pBIN19 Agrobacterium engineering bacteria liquid contaminated 15 minutes, after aseptic thieving paper sucks unnecessary bacterium liquid, access altogether culture medium (contains 3% (w/w) sucrose, 0.8% (w/w) agar, 1mg/L 6-BA and 1mg/L 2, the MS solid medium of 4-D, pH5.8), cultivated altogether 48 hours under 25 ± 2 ℃ of dark conditions, after sterile water wash, change again resistance screening over to and induce the substratum of sprouting (to contain 3% (w/w) sucrose, 0.8% (w/w) agar, 4mg/L 6-BA, 2mg/L ZT, 5mg/L AgNO ₃, 500mg/L Carb and 20mg/L Kan the MS solid medium, pH5.8) in, under 25 ± 2 ℃, photoperiod 16h/d, intensity of illumination 1000-2000lx condition, be cultured to and grow green callus and resistant buds (Figure 13-14); The resistant buds of the long 2-3cm that same vegetative point is grown downcuts, change root media (the 1/2MS solid medium that contains 3% (w/w) sucrose, 0.8% (w/w) agar, 0.2mg/L NAA, 250mg/L Carb and 5mg/L Kan over to, pH5.8) in, under 25 ± 2 ℃, photoperiod 16h/d, intensity of illumination 1000-2000lx condition, be cultured to and take root, namely get transgenosis kale plant (Figure 15).

Above-mentioned agriculture bacillus mediated DWARF, SPWith GA20oxThe method that three gene RNAi carriers transform kale is optimization method, it has following characteristics: 1. concentration affects thalline adhering on vegetable cell of Agrobacterium, within the specific limits, bacterial concentration is larger, adhesive rate is higher, transformation efficiency is larger, but surpass this scope, because the toxic action of bacterium or grow too fast etc., can make explant be subject to excessively injury and to cause brownization of explant otch to be rotted serious of Agrobacterium, and be unfavorable for removing Agrobacterium after the common cultivation, and be lower than this scope, lower bacterial concentration can make T-DNA can not effectively be integrated in the vegetable cell genome, greatly reduces transformation efficiency.The present invention is optimized the concentration of Agrobacterium engineering bacteria liquid, can effectively guarantee BoDWARF, BoSPWith BoGA20oxThe transformation efficiency of three gene RNAi carriers; 2. hormone is one of principal element that affects regeneration frequency, different hormone combinations and hormonal readiness are different with the impact of sprouting on inducing the different explants callus, the hormone combinations of preculture and altogether cultivation is preferably 1mg/L 6-BA and 1mg/L 2 in the inventive method, 4-D, the hormone combinations of differentiation is preferably 4mg/L 6-BA and 2mg/L ZT and adds the short differentiation of 5mg/L AgNO3, and root-promoting hormone is preferably 0.2mg/L NAA; 3. because kale is very sensitive to Kan, and Kan concentration is excessively low, select to press deficiency, easily produce the non-transgenic plant, otherwise then be unfavorable for the bud differentiation, resistance screening induces the concentration of Kan in the substratum of sprouting to be preferably 10-20mg/L in the inventive method; 4. adopt root media of the present invention to carry out root culture, rooting rate can reach 10%, and the positive plant yield can reach 3.6%.

Six, the screening of transgenosis kale positive plant

1.PCR detect the expression of NPTII reporter gene in transgenosis kale plant

NPTII reporter gene sequence according among the carrier pBIN19, design following special primer:

NPTII-F：5'-GACAATCGGCTGCTCTGA-3'(SEQ ID No.13)；

NPTII-R：5'-AACTCCAGCATGAGATCC-3'(SEQ ID No.14)。

Get respectively aforementioned transgenosis kale plant and non-transgenic kale plant, extract genomic dna, again take the gained genomic dna as template, NPTII-F and NPTII-R be as primer carries out pcr amplification, detects the expression of NPTII reporter gene in transgenosis kale plant.The agarose gel electrophoresis analytical results of PCR product as shown in figure 16, the PCR product of part transgenosis kale plant the target dna band occurs in the 875bp position.To be connected with the pMD18-T carrier behind this DNA band recovery purifying, the order-checking proof is consistent with NPTII reporter gene fragment sequence, confirms that these plant are the transgenic positive plant.

2.qPCR detect DWARF, SPWith GA20oxThe expression of three genes in transgenosis kale positive plant

According to kale DWARF, SPWith GA20oxThree gene fragment orders design following specificity qPCR primer and reference gene BoActinThe qPCR primer of (GenBank accession number AF044573):

qBoDWARF-F：5'-GATGCGAGAACGAAGAGAGTC-3'(SEQ ID No.15)；

qBoDWARF-R：5'-TGAAGAAGGAAGATAACCGATAC-3'(SEQ ID No.16)；

qBoSP-F：5'-GACGCCGTTGACTGAAGG-3'(SEQ ID No.17)；

qBoSP-R：5'-CCAGTCATAAGTATCCCG-3'(SEQ ID No.18)；

qBoGA20ox-F：5'-TGAACAGCAAGAGCGAGAGG-3'(SEQ ID No.19)；

qBoGA20ox-R：5'-GTGAACTCAAGCAACATAGACC-3'(SEQ ID No.20)；

qBoactin-F：5'-GGAACTGGAATGGTGAAGGTG-3'(SEQ ID No.21)；

qBoactin-R：5'-GTCTTTCTGACCCATCCCAAC-3'(SEQ ID No.22)。

Get respectively the spire of aforementioned transgenosis kale positive plant and non-transgenic kale plant, extract total RNA, be cDNA with total RNA reverse transcription, again take gained cDNA as template, take qBoDWARF-F and qBoDWARF-R, qBoSP-F and qBoSP-R, qBoGA20ox-F and qBoGA20ox-R, qBoactin-F and qBoactin-R as primer carries out qPCR, detect respectively DWARF, SPWith GA20oxThe expression of three genes in transgenosis kale positive plant.The result as shown in figure 17, in the transgenosis kale positive plant DWARF, SPWith GA20oxTrigenic expression all significantly reduces than non-transgenic kale plant, and gene silencing efficient is high.

Seven, the phenotype analytical of transgenosis kale positive plant

The above-mentioned transgenosis kale positive plant that filters out and non-transgenic kale plant are cultivated according to a conventional method, the phenotypic characteristic of observation analysis transgenosis kale positive plant, the result shows that the strain of transgenosis kale positive plant is microminiaturized phenotype (Figure 18-19).

Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and not depart from the spirit and scope of the present invention that appended claims limits.

＜110〉University Of Chongqing

＜120〉kale DWARF, SPWith GA20oxThree gene RNAi carriers and construction process and application

<160> 22

<210> 1

<211> 974

<212> DNA

＜213〉kale (B Rassica oleraceaL. var. AcephalaDC)

<220>

＜223〉kale DWARFGene fragment

<400> 1

atcgctgcgg ttcacggacc gagccaccgg ctcatgagag gctcactaat ctctctaaca 60

agcccggcta tgatgaagaa cacctctttg cctagattga tgcattcatg agaagttatc 120

tttctggttg ggatgagttc gaaaccgttg acatccaaga aaagaccaaa catatgacat 180

ttttatcatc gttgttacaa atagcagaga ctttgaaaaa accagaggtt gaagaatata 240

aaactgagtt cttcaagctt gttgagggaa ctctttcggt cccaatcgat ctcccaggaa 300

cgcaataccg ttgtggaatc caagcaagaa acaatatcga taggttattg acaaagctga 360

tgcaagaacg aagagagtca ggagaaactt atacagatat gttgggttac ttgatgaaga 420

aggaagataa ccgatacttg ttaactgata aggagataag agatcaagta ttaacgatct 480

tgtattcggg ttatgagact gtctctgtaa cttccatgat ggctcttaag tatctccatg 540

atcaccctaa agctcttgaa gaactcagaa gagaacattt ggctataaga gacagaaaac 600

gacctgacga accgcttaat ctcgacgaca ttaagtccat gaaattcaca cgagctgtga 660

tctttgagac atcaagattg gcaacggttg ttaatggtgt cttgagaaaa actactcacg 720

acttggaact caacgggtat ttaatcccaa aaggttggag aatttacgtc tacacaagag 780

agattaatta tgattcatct ctgtatgaag atccaatgat ctttaaccca tggagatgga 840

tggaaaagaa aatggaatca atgagctatt tcttactttt tggaggtgga gcaagacatt 900

gccctggaaa ggaactagga atttctgaag tctcgagctt cattcactac tttgttacga 960

gatacaaatg ggag 974

<210> 2

<211> 336

<212> DNA

＜213〉kale (B Rassica oleraceaL. var. AcephalaDC)

<220>

＜223〉kale SPGene fragment

<400> 2

cccaaagcag aactaaagca agcccaagct tgtctctccc cattccaaca cacacatttt 60

catatcaaac tcaaaacact atttccttaa tttctttcac ttattcttca tttcttcctc 120

taaattctta accaagcaat cgagatggcc aggatttcct cagacccgct tatggttggg 180

agagtgatcg gagacgttgt ggacaattgt ttgcaggcag tgaaaatgac ggtgacctat 240

aattctgaca agcaagtcta caatggccat gaacttttcc cttctgtagt tacgtacaaa 300

cctaaggttg aagttcatgg gggtgacatg agatca 336

<210> 3

<211> 710

<212> DNA

＜213〉kale (B Rassica oleraceaL. var. AcephalaDC)

<220>

＜223〉kale GA20oxGene fragment

<400> 3

ggtcaatcac ggcatcagcg agcagcttat atctgacgct catgaatata tgtctcgctt 60

cttcgacatg cctctctctg agaaacagag gattcagaga aaagaaggcg agagttgtgg 120

ctacgctagc agtttcactg gccgcttctc caccaagctt ccgtggaagg aaaccctttc 180

tttccagttt tgtgacgaca agagccgccc aaaaaacgtt caagattact tctgcgatgc 240

gttgggacat gagtttgagc catttgggaa agtgtatcaa gagtactgtg aggcaatgag 300

ttctctgtca ctgaagatca tggagctttt ggggataaat ttaggcgtat ccggagacta 360

cttcaaatca ttttttgaag aaaatgattc cataatgaga ctaaattact accctccatg 420

tcaaaaacca gatcttacat ttggaacagg acctcattgt gatcctactt ctcttacaat 480

ccttcaccaa gaccatgttc atggccttca agtctttgtc gataatcaat ggcgctccat 540

tagtcctaac cccaaggcct ttgtggtcaa tatcggtgat actttcatgg ctctatcaaa 600

caatagatac aaaagctgct tgcaccgggc ggtggtgaac agcaagagcg agaggaaatc 660

acttgcgttc ttcctgtgtc ccaaaaaaga cagagtagtg acgccaccga 710

<210> 4

<211> 1997

<212> DNA

＜213〉artificial sequence

<220>

＜223〉kale DWARF/ SP/ GA20oxThree gene splicing fragments

<400> 4

atcgctgcgg ttcacggacc gagccaccgg ctcatgagag gctcactaat ctctctaaca 60

agcccggcta tgatgaagaa cacctctttg cctagattga tgcattcatg agaagttatc 120

tttctggttg ggatgagttc gaaaccgttg acatccaaga aaagaccaaa catatgacat 180

ttttatcatc gttgttacaa atagcagaga ctttgaaaaa accagaggtt gaagaatata 240

aaactgagtt cttcaagctt gttgagggaa ctctttcggt cccaatcgat ctcccaggaa 300

cgcaataccg ttgtggaatc caagcaagaa acaatatcga taggttattg acaaagctga 360

tgcaagaacg aagagagtca ggagaaactt atacagatat gttgggttac ttgatgaaga 420

aggaagataa ccgatacttg ttaactgata aggagataag agatcaagta ttaacgatct 480

tgtattcggg ttatgagact gtctctgtaa cttccatgat ggctcttaag tatctccatg 540

atcaccctaa agctcttgaa gaactcagaa gagaacattt ggctataaga gacagaaaac 600

gacctgacga accgcttaat ctcgacgaca ttaagtccat gaaattcaca cgagctgtga 660

tctttgagac atcaagattg gcaacggttg ttaatggtgt cttgagaaaa actactcacg 720

acttggaact caacgggtat ttaatcccaa aaggttggag aatttacgtc tacacaagag 780

agattaatta tgattcatct ctgtatgaag atccaatgat ctttaaccca tggagatgga 840

tggaaaagaa aatggaatca atgagctatt tcttactttt tggaggtgga gcaagacatt 900

gccctggaaa ggaactagga atttctgaag tctcgacgat tcccaaagca gaactaaagc 960

aagcccaagc ttgtctctcc ccattccaac acacacattt tcatatcaaa ctcaaaacac 1020

tatttcctta atttctttca cttattcttc atttcttcct ctaaattctt aaccaagcaa 1080

tcgagatggc caggatttcc tcagacccgc ttatggttgg gagagtgatc ggagacgttg 1140

tggacaattg tttgcaggca gtgaaaatga cggtgaccta taattctgac aagcaagtct 1200

acaatggcca tgaacttttc ccttctgtag ttacgtacaa acctaaggtt gaagttcatg 1260

ggggtgacat gagatcatct agtgatttcg gtggcgtcac tactctgtct tttttgggac 1320

acaggaagaa cgcaagtgat ttcctctcgc tcttgctgtt caccaccgcc cggtgcaagc 1380

agcttttgta tctattgttt gatagagcca tgaaagtatc accgatattg accacaaagg 1440

ccttggggtt aggactaatg gagcgccatt gattatcgac aaagacttga aggccatgaa 1500

catggtcttg gtgaaggatt gtaagagaag taggatcaca atgaggtcct gttccaaatg 1560

taagatctgg tttttgacat ggagggtagt aatttagtct cattatggaa tcattttctt 1620

caaaaaatga tttgaagtag tctccggata cgcctaaatt tatccccaaa agctccatga 1680

tcttcagtga cagagaactc attgcctcac agtactcttg atacactttc ccaaatggct 1740

caaactcatg tcccaacgca tcgcagaagt aatcttgaac gttttttggg cggctcttgt 1800

cgtcacaaaa ctggaaagaa agggtttcct tccacggaag cttggtggag aagcggccag 1860

tgaaactgct agcgtagcca caactctcgc cttcttttct ctgaatcctc tgtttctcag 1920

agagaggcat gtcgaagaag cgagacatat attcatgagc gtcagatata agctgctcgc 1980

tgatgccgtg attgacc 1997

<210> 5

<211> 17

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer BoDF-F

<400> 5

atcgctgcgg ttcacgg 17

<210> 6

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer BoDF-R

<400> 6

ctcccatttg tatctcgta 19

<210> 7

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer BoSP-F

<400> 7

cccaaagcag aactaaagc 19

<210> 8

<211> 17

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer BoSP-R

<400> 8

tgatctcatg tcacccc 17

<210> 9

<211> 18

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer BoGA20ox-F

<400> 9

ggtcaatcac ggcatcag 18

<210> 10

<211> 18

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer BoGA20ox-R

<400> 10

tcggtggcgt cactactc 18

<210> 11

<211> 32

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer DWARF (F+EX)

<400> 11

ccggaattct ctagaatcgc tgcggttcac gg 32

<210> 12

<211> 33

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer GA20ox (F+KB)

<400> 12

cggggtaccg gatccggtca atcacggcat cag 33

<210> 13

<211> 18

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer NPTII-F

<400> 13

gacaatcggc tgctctga 18

<210> 14

<211> 18

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer NPTII-R

<400> 14

aactccagca tgagatcc 18

<210> 15

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer qBoDWARF-F

<400> 15

gatgcgagaa cgaagagagt c 21

<210> 16

<211> 23

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer qBoDWARF-R

<400> 16

tgaagaagga agataaccga tac 23

<210> 17

<211> 18

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer qBoSP-F

<400> 17

gacgccgttg actgaagg 18

<210> 18

<211> 18

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer qBoSP-R

<400> 18

ccagtcataa gtatcccg 18

<210> 19

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer qBoGA20ox-F

<400> 19

tgaacagcaa gagcgagagg 20

<210> 20

<211> 22

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer qBoGA20ox-R

<400> 20

gtgaactcaa gcaacataga cc 22

<210> 21

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer qBoactin-F

<400> 21

ggaactggaa tggtgaaggt g 21

<210> 22

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉primer qBoactin-R

<400> 22

gtctttctga cccatcccaa c 21

Claims (10)

1. kale DWARFGene fragment is characterized in that, nucleotide sequence is shown among the SEQ ID No.1 the 1st to the 936th.

2. kale SPGene fragment is characterized in that, nucleotide sequence is shown in SEQ ID No.2.

3. kale GA20oxGene fragment is characterized in that, nucleotide sequence is shown in SEQ ID No.3.

4. kale DWARF, SPWith GA20oxThree gene RNAi carriers is characterized in that, contain a hpRNA expression cassette, described hpRNA expression cassette comprise the promotor that is linked in sequence, DWARF/ SP/ GA20oxThe forward fragment of three gene splicing fragments, DWARF/ SP/ GA20oxReverse fragment and the terminator of three gene splicing fragments; Described DWARF/ SP/ GA20oxThree gene splicing fragments comprise DWARFGene fragment or its complementary fragment, SPGene fragment or its complementary fragment and GA20oxGene fragment or its complementary fragment, described DWARFThe nucleotide sequence of gene fragment shown among the SEQ ID No.1 the 1st to the 936th, SPThe nucleotide sequence of gene fragment shown in SEQ ID No.2, GA20oxThe nucleotide sequence of gene fragment is shown in SEQ ID No.3.

5. kale according to claim 4 DWARF, SPWith GA20oxThree gene RNAi carriers is characterized in that, described hpRNA expression cassette comprise the CaMV35S promotor that is linked in sequence, DWARF/ SP/ GA20oxThe forward fragment of three gene splicing fragments, PDK intron, DWARF/ SP/ GA20oxThe reverse fragment of three gene splicing fragments and Ocs terminator; Described DWARF/ SP/ GA20oxThree gene splicing fragments comprise and being linked in sequence DWARFThe forward fragment of gene fragment, SPThe forward fragment of gene fragment and GA20oxThe reverse complemental fragment of gene fragment.

6. kale according to claim 5 DWARF, SPWith GA20oxThree gene RNAi carriers is characterized in that, take the pBIN19 carrier as skeleton, described hpRNA expression cassette inserts in the multiple clone site of pBIN19 carrier, and is described DWARF/ SP/ GA20oxThe nucleotide sequence of three gene splicing fragments is shown in SEQ ID No.4.

7. each described kale of claim 4 to 6 DWARF, SPWith GA20oxThree gene RNAi Vector construction methods is characterized in that, may further comprise the steps:

A. kale DWARF, SPWith GA20oxThe clone of three gene fragments: take the total RNA of kale spire as template, reverse transcription obtains cDNA, again take gained cDNA as template, respectively take BoDF-F and BoDF-R, BoSP-F and BoSP-R, BoGA20ox-F and BoGA20ox-R as primer, the pcr amplification nucleotide sequence is shown in SEQ ID No.1 DWARFGene fragment, nucleotide sequence are shown in SEQ ID No.2 SPGene fragment and nucleotide sequence are shown in the SEQ ID No.3 GA20oxGene fragment; The nucleotide sequence of described primer BoDF-F and BoDF-R is respectively shown in SEQ ID No.5 and SEQ ID No.6, shown in SEQ ID No.7 and SEQ ID No.8, the nucleotide sequence of BoGA20ox-F and BoGA20ox-R is respectively shown in SEQ ID No.9 and SEQ ID No.10 respectively for the nucleotide sequence of BoSP-F and BoSP-R; Then, with the pcr amplification gained DWARFGene fragment and SPGene fragment is connected with the pMD18-T carrier respectively, obtains recombinant plasmid DWARF::pMD18-T and SP::pMD18-T, in addition with the pcr amplification gained GA20oxGene fragment is connected with pGEM-T Easy carrier, obtains recombinant plasmid GA20ox::pGEM-T Easy;

B. kale DWARF, SPWith GA20oxThe splicing of three gene fragments: recombinant plasmid DWARF::pMD18-T is used XhoI and EcoThe RI double digestion reclaims purification of Recombinant plasmid DWARF::pMD18-T linear fragment, in addition recombinant plasmid SP::pMD18-T is used SalI and EcoThe RI double digestion reclaims purifying SPGene fragment, again with the recombinant plasmid DWARF::pMD18-T linear fragment of purifying with SPGene fragment connects, and obtains recombinant plasmid SP/DF::pMD18-T; Then, recombinant plasmid SP/DF::pMD18-T is used XbaI and EcoThe RI double digestion reclaims purification of Recombinant plasmid SP/DF::pMD18-T linear fragment, in addition recombinant plasmid GA20ox::pGEM-T Easy is used SpeI and EcoThe RI double digestion reclaims purifying GA20ox gene fragment, and the recombinant plasmid SP/DF::pMD18-T linear fragment with purifying is connected with the GA20ox gene fragment again, obtains recombinant plasmid SP/DF/GA20ox::pMD18-T;

C. kale DWARF, SPWith GA20oxThree gene RNAi Vector constructions: take recombinant plasmid SP/DF/GA20ox::pMD18-T as template, take DWARF (F+EX) and GA20ox (F+KB) as primer, pcr amplification both sides band restriction enzyme site DWARF/ SP/ GA20oxThree gene splicing fragments; The nucleotide sequence of described primer DWARF (F+EX) and GA20ox (F+KB) is respectively shown in SEQ ID No.11 and SEQ ID No.12; The PCR product of purifying is used XbaI and BamThe HI double digestion reclaims purifying DWARF/ SP/ GA20oxThree gene splicing fragments, the pHANNIBAL carrier with the same double digestion of warp is connected again, obtains recombinant plasmid SP/DF/GA20ox::pHANNIBAL; In addition the PCR product of purifying is used KpnI and EcoThe RI double digestion reclaims purifying DWARF/ SP/ GA20oxThree gene splicing fragments, the recombinant plasmid SP/DF/GA20ox::pHANNIBAL with the same double digestion of warp is connected again, obtains to contain recombinant plasmid (SP/DF/GA20ox) i::pHANNIBAL of hpRNA expression cassette; Then, recombinant plasmid (SP/DF/GA20ox) i::pHANNIBAL is used SpeI and SacThe I double digestion reclaims purifying hpRNA expression cassette, in addition plant expression vector pBIN19 is used XbaI and SacThe I double digestion reclaims cmy vector pBIN19 linear fragment, and the carrier pBIN19 linear fragment with purifying is connected with the hpRNA expression cassette again, obtains recombinant plasmid (SP/DF/GA20ox) i::pBIN19, i.e. kale DWARF, SPWith GA20oxThree gene RNAi carriers.

8. contain each described kale of claim 4 to 6 DWARF, SPWith GA20oxThe microbial transformant of three gene RNAi carriers.

9. each described kale of claim 4 to 6 DWARF, SPWith GA20oxThe application of three gene RNAi carriers in the breeding of microminiaturized kale plant.

10. utilize each described kale of claim 4 to 6 DWARF, SPWith GA20oxThree gene RNAi carriers obtain the method for microminiaturized kale plant, it is characterized in that, may further comprise the steps:

A. the preparation of kale explant: after the kale seed disinfection, be seeded in that to contain massfraction be that 3% sucrose and massfraction are that 0.8% agar, pH are on 5.8 the 1/2MS solid medium, under 25 ± 2 ℃, photoperiod 16h/d, intensity of illumination 1000-2000lx condition, cultivate; 7-10 days Bands handle cotyledon of growth is downcut, it is that 3% sucrose, massfraction are 0.8% agar, 1mg/L 6-benzyl aminoadenine and 1mg/L 2 that access contains massfraction, 4-D, pH are in 5.8 the MS solid medium, preculture is 48 hours under 25 ± 2 ℃ of dark conditions, makes the kale explant;

B. the preparation of Agrobacterium engineering bacteria liquid: will contain kale DWARF, SPWith GA20oxThe Agrobacterium engineering strain of three gene RNAi carriers is inoculated in that to contain massfraction be 1.2% agar, the 50mg/L Rifampin, 500mg/L Streptomycin sulphate and 50mg/L kantlex, pH is on 7.2 the YEB solid medium, activate 2-3 days to growing single bacterium colony under 28 ± 2 ℃ of dark conditions, picking list bacterium colony, with containing the 50mg/L Rifampin, 500mg/L Streptomycin sulphate and 50mg/L kantlex, pH is 7.2 YEB liquid nutrient medium 20mL, at 28 ± 2 ℃, enlarged culturing is 1.5 days under the 200rpm condition, again gained bacterium liquid is contained the 50mg/L Rifampin for the 1:100 access by volume, 500mg/L Streptomycin sulphate and 50mg/L kantlex, pH is in 7.2 the YEB liquid nutrient medium, at 28 ± 2 ℃, enlarged culturing is to OD under the 200rpm condition ₆₀₀=1.8-2.0,28 ± 2 ℃ of centrifugal supernatants of abandoning then, thalline be with fresh YEB liquid nutrient medium washing, is that 3% sucrose, pH are that 5.8 MS salts solution 100mL is resuspended with containing massfraction again, makes Agrobacterium engineering bacteria liquid;

C. Agrobacterium infect conversion: after the kale explant that step a is made is put and is contaminated 15 minutes in the Agrobacterium engineering bacteria liquid that step b makes, it is 3% sucrose that access contains massfraction, massfraction is 0.8% agar, 1mg/L 6-benzyl aminoadenine and 1mg/L 2,4-D, pH is in 5.8 the MS solid medium, cultivated altogether 48 hours under 25 ± 2 ℃ of dark conditions, change again that to contain massfraction be 3% sucrose over to, massfraction is 0.8% agar, 4mg/L 6-benzyl aminoadenine, the 2mg/L zeatin, the 5mg/L Silver Nitrate, 500mg/L Pyocianil and 20mg/L kantlex, pH is in 5.8 the MS solid medium, at 25 ± 2 ℃, photoperiod 16h/d, be cultured under the intensity of illumination 1000-2000lx condition and grow callus and resistant buds; The resistant buds of long 2-3cm is downcut, change that to contain massfraction be that 3% sucrose, massfraction are that 0.8% agar, 0.2mg/L naphthylacetic acid, 250mg/L Pyocianil and 5mg/L kantlex, pH are in 5.8 the 1/2MS solid medium over to, under 25 ± 2 ℃, photoperiod 16h/d, intensity of illumination 1000-2000lx condition, be cultured to and take root, namely get transgenosis kale plant;

D. the acquisition of transgenic positive plant: detect kale by PCR DWARF, SPWith GA20oxThe expression of the reporter gene that contains in the three gene RNAi carriers in transgenosis kale plant, and qPCR detects DWARF, SPWith GA20oxThe expression of three genes in transgenosis kale plant filters out the transgenic positive plant from the transgenosis kale plant that step c obtains, namely obtain microminiaturized kale plant.

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