CN102212523A - DNA molecule for expressing hairpin RNA for inhibiting wheat starch branching enzyme IIa (SBEIIa) and application thereof - Google Patents

Abstract

The invention discloses a DNA molecule for expressing hairpin RNA for inhibiting wheat starch branching enzyme IIa (SBEIIa) and application thereof. The invention provides an RNA molecule, the nucleotide sequence of which is the sequence 4 in a sequence table. The invention also provides a DNA molecule for expressing the RNA, wherein the nucleotide sequence of the DNA molecule is the sequence 5 in the sequence table. Proved by experiments, positive and negative RNA interfering vectors inserted with wheat grain SBEIIa gene fragments are obtained by using an RNA interference (RNAi) technology; and when the RNA interfering vectors are transferred to wheat, the amylose content of wheat grain starch is improved and the quality of the wheat starch is improved on the premise that vegetative growth and stress resistance of the wheat are not affected.

Description

The dna molecular and the application thereof of the hairpin RNA of expression inhibiting wheat SBEIIa

Technical field

The present invention relates to biological technical field, relate in particular to the dna molecular and the application thereof of the hairpin RNA of a kind of expression inhibiting wheat SBEIIa.

Background technology

Amylose content generally accounts for about 25% of common wheat starch, accounts for about 12～16% of whole meal flour.The amylose starch specific molecular structure has caused its distinctive physico-chemical property, processing characteristics and using value: as 1) because the amylose starch good film-forming property is the important source material of producing edible food package material, film etc.; 2) because amylose starch belongs to resistant starch (resistant starch), be difficult for being digested by the enteron aisle enzyme liberating, edible back human body glucose level does not have significantly and raises, and is used for preparation to diabetics's special food, solves patient's hunger sensation; Has the good health care prophylactic effect for obesity, gallstone disease, intestines dysfunction and colorectal carcinoma etc.; As functional foodstuff low in calories, can promote mineral substance absorption, reducing cholesterol and management of body weight; 3) still produce the necessary material of environment-friendly type degradable film, industrial spirit, purposes is very extensive.

It is very high to extract the amylose starch cost from (wheat, corn, potato etc.) common starch.Therefore, China's amylose starch mainly relies on import.Because 1) China's wheat is to be used for preparing traditional flour food more than 90%, processing qualities such as the ductility of whole meal flour are better than other crop flour, like this, the whole meal flour of high-content amylose starch, be suitable for directly making diabetics's various flour food, protective foods, thereby when enjoying the original delicious food of food, obtain health and nutrition; 2) adjust the wheat breed structure, solve the superfluous relatively problem of wheat yield (refer to high-quality, wheat special deficiency, and inferior, common wheat is superfluous); Therefore, the wheat breed of cultivation high-content amylose starch is significant.But domestic still do not have the wheat mutative material that screens high amylose starch at present, only has Japan's report to screen amylosynthease (SGP-1) mutant (its amylose content also only reaches about 37%) abroad, limited the breed of variety of high amylose starch wheat.

In recent years, the develop rapidly of Agricultural biotechnologies was that the genetic improvement that utilizes the means of genetic engineering to carry out wheat starch has been opened up wide prospect.(2005) such as (1994), Li Jiarui such as Dong Yunzhou (1993), Gerhard etc. (2000) and Rahman have been studied genetic manipulation respectively and have been changed the influence of potato and wheat starch route of synthesis, thereby change the starch The Nomenclature Composition and Structure of Complexes.

Q-enzyme (SBE) hydrolysis long-chain non reducing end α-1, the 4-glycosidic link, downcut an oligose fragment, again this fragment is transferred in the starch sugar chain on the glucosyl residue, generate α-1, the 6-glycosidic link, the branched structure of formation amylopectin, branch can continue to extend under the amylosynthease effect.Pea SBEa that Burton etc. (1995) obtain separation and the cDNA of SBEb, the SBE sequence of same corn, paddy rice, potato etc. relatively after, SBE is divided into two big class: SBEI and SBEII.The substrate of SBEI effect is an amylose starch, and it can make long glycosidic link transfer on the amylose starch; And the substrate of SBEII effect is an amylopectin, can make short glycosidic link transfer to (Taketa etc., 1993 on the amylopectin; Morell etc., 1997).In wheat endosperm development process, the peak expression of SBEII will be early than SBEI, and SBEII is spending back 5d to begin to express, and spends back 10d just can reach the highest; And SBEI is spending back 10d to begin to express, and expresses behind the 5d just to reach the highest (Morell etc., 1997; Kent, 2002).To in finding after corn, potato and the wheat research, the reduction alive of SBEI enzyme can't influence the content of amylose starch respectively for Gao (1997), Safford (1998) and Regina (2004).

The cDNA of coding SBEIIa and SBEIIb is by clone, location in crops such as corn, paddy rice, wheat.In Different Crop, SbeIIa and SbeIIb relative expression level are different in the different tissues organ.In monocotyledons, SBEIIa is expressed in the nutritive issues such as embryo, leaf and other, and SBEIIb is then single-minded to be expressed in (Fisher, Gao, 1997) in embryo and the endosperm.At corn, SbeIIa expression level in nutritive issue is higher, and a small amount of expression is arranged in endosperm, and SbeIIb is high level expression (Gao, 1997) in endosperm only.

Summary of the invention

An object of the present invention is to provide a kind of RNA molecule.

RNA molecule provided by the invention, its nucleotides sequence are classified the sequence 4 in the sequence table as.

Another object of the present invention provides a kind of dna molecular of expressing described RNA.

Dna molecular provided by the invention, its nucleotides sequence are classified the sequence 5 in the sequence table as.

Described dna molecular is connected to form successively by A, B and three fragments of C, and the segmental nucleotides sequence of described A is classified the sequence 1 in the sequence table as, described C fragment and described A fragment reverse complemental; The segmental nucleotides sequence of described B is classified the sequence 2 in the sequence table as.

The recombinant expression vector, transgenic cell line, reorganization bacterium or the expression cassette that contain described dna molecular also are the scope of protection of the invention.

The carrier that described recombinant expression vector obtains for the XmaI restriction enzyme site that described dna molecular is inserted the pBAC47P carrier

The described dna molecular total length that increases and any segmental primer thereof are to also being the scope of protection of the invention, and a primer of the segmental primer centering of the A in the wherein said dna molecular is the sequence 4 in the sequence table, and another primer is the sequence 5 in the sequence table.

The application in the quality of improvement wheat starch of described RNA or described dna molecular also is a scope of protection of the invention; The quality of described wheat starch is for embodying by the wheat grain amylose content.

The 3rd purpose of the present invention provides a kind of transgenic plant method of cultivating high amylose content.

Method provided by the invention is with the forfeiture of the SBEIIa gene function in the purpose plant, obtains transgenic plant, and the amylose content in the seed of described transgenic plant is higher than described purpose plant.

Described SBEIIa gene function in purpose plant forfeiture will be realized by will the described dna molecular of claim 2 importing in the purpose plant.

Described dna molecular imports by described recombinant expression vector;

Described purpose plant is monocotyledons or dicotyledons, and described monocotyledons is a wheat;

The nucleotides sequence of described SBEIIa gene is classified the sequence 1 in the sequence table as.

Of the present invention experimental results show that, utilize the RNAi technology to obtain inserting wheat grain SbeIIa gene fragment forward and reverse rna interference vector, change in the wheat, do not influence that wheat is nourished and grown and the prerequisite of resistance under improve the amylose content of wheat grain starch, the quality of improvement wheat starch.Therefore, to enlarging the application of wheat starch in food and non-food product industry, have important theory and practice significance.This research will improve for wheat starch, quality breeding provides theoretical foundation, experimental technique and breeding material, and will be significant to China's Wheat Quality Improvement and fine quality seed selection.

Description of drawings

Fig. 1 is a wheat grain filling seed total RNA in mid-term

Fig. 2 obtains SbeIIa fragment (about 1kb) for RT-PCR

Fig. 3 is an expression vector pBAC47P+sbeIR structural representation

Fig. 4 is T ₀For transfer-gen plant Intron primer PCR detected result

Fig. 5 is T ₀For transfer-gen plant SBEIIa primer PCR detected result

Fig. 6 is T ₀In generation, changes SbeIIa gene plant PCR-Southern and analyzes

Fig. 7 is T ₁Analyze for transgenic wheat sxemiquantitative RT-PCR

Fig. 8 is T ₁For the transgenic wheat strain is sxemiquantitative RT-PCR analytical results

Fig. 9 is T ₅For the transgenic wheat strain is the percentage composition of amylose starch in the flour

Embodiment

Employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

The acquisition of embodiment 1, rna interference vector

1, the SbeIIa gene fragment obtains

By wheat SBE encoding gene mRNA homology relatively, GenBank search wheat SbeIIa gene mRNA sequence (GenBank NO.:Y11282, AF338431), use Primer5.0 software design primer to be:

SBEIIa-s2:5 '-CGAGATGGTGGCTTGAAGAA-3 ' (sequence 4)

SBEIIa-a2:5 '-ATGATCAAGCCTGCTGAATCC-3 ' (sequence 5)

(Triticum aestivum L, this material " often becomes Zhang Haiping, You Mingshan, Li Baoyun, Li Weihua, Liu Guangtian at document to " agricultural university 152 " wheat that adopts Trizol RNA to extract test kit (day root company) to extract pollination 15d.Xitix is to the research of wheat dough sheet color and luster influence.China's grain and oil journal, 2006,21 (6): 27-30 " disclosed in; the public can obtain from China Agricultural University) total RNA of seed (Fig. 1; 1,2,3,4 RNA that are extraction wherein); the two-step approach with M-MLV reverse transcription test kit (Promega company) by specification is carried out reverse transcription-PCR (RT-PCR), obtains the PCR product (Fig. 2 wherein 1,2 is the PCR product) about 980bp.Reclaim the PCR product, use pGEM-T Easy Vector System (Promega company) to specifications this PCR product to be connected on the pGEM-TEasy carrier, to connect product thermal shock transformed competence colibacillus EcoliDH5 α, obtain transformant, extract the plasmid of transformant, this plasmid is for inserting the carrier that obtains among the pGEM-T Easy with the sequence in the sequence table 1, sequence 1 in the sequence table reaches 98.9% with the Y11282mRNA homology of GenBank, the SbeIIa gene that this PCR product is a wheat is described, with this plasmid called after pTE-sbe.

2, the SbeIIa fragment is oppositely inserted the downstream of FAD2Intron1

Cut the pTE-sbe and the pBSK-FAD2Int1 of above-mentioned acquisition with restriction endonuclease EcoRI while enzyme, reclaim the former the about 1kb fragment and the latter's big fragment behind the agarose gel electrophoresis respectively, connect test kit (TakaRa company) with T4DNA this two fragments connection is obtained connecting product, transformed competence colibacillus E.coliDH5 α bacterial strain, obtain transformant, extract the plasmid of transformant, carry out pcr amplification with primer SBEIIa-s2 and AtFAD2Int1F (5 '-AGCAGATCTATCGTGAGCGG-3 '), amplify about 1.0KB positive plasmid of segmental plasmid of (containing the about 100bp fragment in reverse SbeIIa gene fragment and FAD2Intron1 downstream), this plasmid is sent to order-checking, the carrier that the result obtains for the EcoRI restriction enzyme site that the sequence in the sequence table 1 (SbeIIa gene fragment) is inserted pBSK-FAD2Int1, and (nucleotides sequence of FAD2Intron1 is classified the sequence 2 in the sequence table as for the FAD2Intron1 that oppositely inserts pBSK-FAD2Int1, with GenBank NO:AJ271841.1 sequence reverse complemental) downstream, with this plasmid called after pBSK-FAD2I1-sbe.

PBSK-FAD2Int1 is prepared as follows: according to AJ271841.1 sequences Design primers F the ADI-5:5 '-GGTA among the GenBank AATTCAGATC

-3 ' (5 ' adds KpnI, Xma I and EcoR I restriction enzyme site successively, protection base AGATC) and FADI-3:5 '-

CGGCCGCAAGCTT

-3 ' (5 ' adds Sac I successively; Xma I and Not I restriction enzyme site; protection base AAGCTT); with the Arabidopis thaliana Columbia ecotype (Arabidopsis thaliana (L.); this material is at document " Zhongfu Ni; Eun-Deok Kim; Misook Ha; Erika Lackey; Jianxin Liu; Yirong Zhang; Qixin Sun; Z.Jeffrey Chen.Altered circadian rhythms regulate growth vigour in hybrids and allopolyploids.Nature; 457 (7227): disclosed among the 327-331 (15January2009), the public can obtain from China Agricultural University) genomic dna be that template is carried out PCR reaction, the PCR product that obtains by Kpn I and Sac I double digestion rear clone to German Stratagene company

II XR Predigested Vector obtains pBSK-FAD2Int1.

3, SbeIIa fragment forward inserts the upstream of FAD2 Intron1

Cut pTE-sbe and pBSK-FAD2I1-sbe with restriction endonuclease NotI while enzyme, reclaim the former the about 1kb fragment and the latter's big fragment behind the agarose gel electrophoresis respectively, with T4DNA enzyme (TakaRa company) this two fragments connection is obtained connecting product, transformed competence colibacillus E.coli DH5 α, obtain transformant, extract plasmid, cut (owing near the segmental 0.735kb of this sbeIIa an EcoRV restriction enzyme site is arranged with the EcoRV enzyme, so available EcoRV enzyme is cut the direction of insertion that detects just arm:, then can obtain the 2.1kb fragment if just arm is identical with antisense arm direction of insertion; If just arm is opposite with antisense arm direction of insertion, then can obtain the 1.63kb fragment), wherein, the EcoRV enzyme is cut and is obtained the carrier that the segmental plasmid of 1.63kb obtains for the NotI place that the sequence in the sequence table 1 (SbeIIa gene) is inserted pBSK-FAD2I1-sbe, and the SbeIIa gene is the FAD2Intron1 upstream that forward inserts pBSK-FAD2I1-sbe, this plasmid called after pBSK-sbeIR.The SbeIIa gene (sequence 1) that forward wherein inserts, FAD2Intron1 (sequence 2) and reverse insertion SbeIIa gene (the reverse complemental chain of sequence 1) have been formed the linear DNA (nucleotides sequence of this dna molecular is classified the sequence 6 in the sequence table as, and the nucleotides sequence of the RNA of its expression is classified the sequence 6 in the sequence table as) of the segmental hairpin RNA of SbeIIa.

4, be inserted into 1Dx5 promotor and enhanser downstream thereof

Cut pBAC47P and pBSK-sbeIR (pBSK-sbeIR was not activated son originally) with restriction endonuclease XmaI while enzyme, reclaim big fragment (the big fragment of segmental carrier that contains 1Dx5 promotor and enhanser thereof of the former 3.5KB behind the agarose gel electrophoresis respectively, 1Dx5 promotor and enhanser thereof get the big or small 0.9KB of being about, its nucleotides sequence is classified sequence 3 as) and latter 3.0kb (linear DNA of the segmental hairpin RNA of SbeIIa) fragment, with the T4DNA ligase enzyme this two fragment is connected, connect product transformed competence colibacillus E.coli DH5 α bacterial strain, obtain transformant, extract the plasmid of transformant, send to order-checking, the result is with the sequence in the sequence table 1, the carrier that the XmaI restriction enzyme site of sequence (linear DNA of the segmental hairpin RNA of SbeIIa) the insertion pBAC47P that the reverse complementary sequence of sequence 2 and sequence 1 is formed obtains, with this carrier called after pBAC47P+sbeIR (Fig. 3), this carrier contains by 1Dx5 promotor and enhanser (sequence 3) thereof, the SbeIIa (sequence 1) that forward inserts, the dna molecular of the inhibition hairpin RNA that FAD2Intron1 (sequence 2) and the SbeIIa (reverse complementary sequence of sequence 1) that oppositely inserts form, this dna molecular called after sbeIR.

PBAC47P obtains by the following method: the pSP72 carrier with Promega company is the carrier that sets out, between HindIII and Sal I, insert the promotor of wheat high-molecular-weight glutelin subunit 1Dx5 and contain enhancer sequence (sequence 3), between Kpn I and EcoR I, insert NOS terminator (GenBank NO:AF485783.1).The about 3.5KB of pBAC47P plasmid size.The NOS terminator comes from the pBI121 carrier of U.S. Clontech company.1Dx5 promotor and contain enhancer sequence and derive from plant expression vector pBPC30.Plant expression vector pBPC30 is at document " Zhang Xiaodong, Liang Rongqi, Chen Xuqing, Yang Fengping, Zhang Liquan.The acquisition of high-quality HMW gluten subunit transgenic wheat and genetic stability thereof and quality trait analysis.Science Bulletin, 2003,48 (5): 474-479 " disclosed in, the public can obtain from China Agricultural University.

Embodiment 2, commentaries on classics sbeIR wheat obtain and Function Identification

One, changes acquisition of sbeIR wheat and evaluation

Acceptor: the common wheat kind is protected the wheat commercial variety that rich 104 (Triticum aestivum L) are the Tianjin authorizations of Plant Protection institute, Chinese Academy of Agricultral Sciences's Powdery Mildew group cultivation, document " Zhou Yilin, Duan Xiayu, Sheng Baoqin, the power people of department.The high-yield disease resisting New Winter Wheat Variety---protect rich 104.The wheat crops journal, 2003,23 (4): 147 " disclosed in, the public can obtain from China Agricultural University, hereinafter to be referred as the wild-type wheat.

Explant: wheat immature embryo is induced the callus that obtains;

Bacterial strain: intestinal bacteria (Escherichia coli) bacterial strain DH5 α;

Carrier: the pBAC47P+sbeIR that obtains by embodiment 1, (the pSP72 carrier with Promega company is the carrier that sets out to pBAC35SIH3, between HindIII and Sal I, insert CaMV 35S promoter (GenBank NO:AF485783.1), between Kpn I and EcoR I, insert NOS terminator (GenBank NO:AF485783.1), insert bar gene (GenBank NO:AF013602.1) at BamHI and Sac I restriction enzyme site.CaMV 35S promoter and NOS terminator come from the pBI121 carrier of U.S. Clontech company.The encoding gene bar of antiweed PPT derives from plant expression vector pBPC30.

Substratum:

(a) rataria callus of induce substratum MS+2,4-D 2mg/L pH5.8

(b) screening culture medium MS+Basta 0.2mL/L pH5.8

(c) the anti-zeatin 10mg/L+Basta of division culture medium MS+IAA 1mg/L+ 0.05mL/L pH5.8

(d) strengthening seedling and rooting substratum MS+IAA 10mg/L+PBA 5mg/L, sucrose 80g/L, agar powder 6g/L, pH5.8

1, changeing the sbeIR wheat obtains

The wild-type wheat children tassel of choosing the back 12～15d of blooming strips seed, strips rataria after the sterilization, and scultellum upwards places on the callus inducing medium, 25 ± 1 ℃ of dark 3～5d inducing wheat callus of cultivating, the callus of acquisition color cadmium yellow.Select the good callus lines of growth conditions to carry out particle gun cotransformation (pBAC47P+sbeIR: pBAC35SIH3=3: 1 (mol ratio also is a molecule number ratio)); Move to behind 25 ± 1 ℃ of dark cultivation 2～3d of callus after the conversion and carry out the bar resistance screening on the screening culture medium; The callus that has the callus lines president of resistance to make new advances during the screening, nonresistant callus then browning is died; Observe after 1～2 week of screening, growth conditions is good, color cadmium yellow callus transfer to and make its differentiation on the division culture medium, the sign that differentiation is arranged on the visible callus piece about 10d, about 4～5 Zhou Houke differentiate seedling, divide individual plant to transfer to carry out strong plantlets and rootage in the strong plantlets and rootage substratum that (5～7d can observe root system and grow, continue to cultivate for 3～4 weeks, in great numbers up to root growth, the green seedling of wheat is enough sturdy; Transplant to soil behind hardening 2～3d, obtain 166 strain T ₀In generation, changeed the sbeIR stem and leaf of Wheat.

2, change the evaluation of sbeIR wheat

1) PCR identifies

A, extraction T ₀In generation, changeed the genomic dna of sbeIR stem and leaf of Wheat, with FAD2Intron1 fragment upstream primer IntronF and downstream primer IntronR is primer, carry out pcr amplification, primer: IntronF:5 '-CCGCTCACGATAGATCTGCT-3 ', IntronR:5 '-GGTAACGATTCAAGAGAGTCTTC-3 ', the result as shown in Figure 4, M:15000bp Marker; +: positive control pBAC47P+sbeIR;-: negative control (wild-type wheat); 0: the water contrast; Swimming lane 1-11: be T ₀In generation, changeed the sbeIR wheat, if obtain the then positive T of 1.0KB fragment ₀In generation, changeed the sbeIR wheat.

B, extraction T ₀In generation, changeed the genomic dna of sbeIR wheat, is that primer increases with SBEIIa-s2 and SBEIIa-a2, the result as shown in Figure 5, wherein, M:DL2000bp Marker; Positive control pBAC47P+sbeIR;-: negative control (wild-type wheat); 0: the water contrast; Swimming lane 1-6: be T ₀In generation, changeed the sbeIR wheat, if obtain the then positive T of 980bp left and right sides fragment ₀In generation, changeed the sbeIR wheat.

Therefore will identify that being the male note identifies positive T as PCR through above-mentioned FAD2 Intronl PCR (A) and SBEIIa PCR (B) ₀In generation, changeed the sbeIR wheat.

2) the PCR-Southern blotting of transfer-gen plant

Extract PCR and identify positive T ₀In generation, changeed the genomic dna of sbeIR wheat, and the 1.0KB PCR product that uses the primer among dNTP (wherein labelled with radioisotope dGTP) and the above-mentioned A to obtain is probe, the result as shown in Figure 6 ,+: positive control pBAC47P+sbeIR; 0: the water contrast; Swimming lane 1-20: for PCR identifies positive T ₀In generation, changeed the sbeIR wheat, and as seen from the figure, what hybridization signal occurs at the 1.0KB place identifies positive T for Southern ₀In generation, changeed the sbeIR wheat, and negative control does not have hybrid belt, and further proof has been integrated the FAD2Intron1 external source fragment on the RNAi carrier in the genome of part transgenic wheat plant.

Be accredited as male transfer-gen plant note with above-mentioned two kinds and make positive T ₀In generation, changeed the sbeIR wheat, obtains the positive T of 24 strains altogether ₀In generation, changeed the sbeIR wheat.

From positive T ₀In generation, changeed sbeIR harvesting wheat seed, and sowing obtains T1 for changeing the sbeIR wheat, adopts above-mentioned 1) and 2) authentication method, obtain the positive T1 of 30 strains for commentaries on classics sbeIR wheat.

3) RT-PCR of transfer-gen plant identifies

Extract the positive T1 of 30 strains for the RNA that changes the sbeIR wheat, reverse transcription obtains RNA, and the cDNA sequences Design gene-specific primer according to the SbeIIa gene carries out RT-PCR, and primer sequence is as follows:

Sbe-2a-a2：5’-CGAGATGGTGGCTTGAAGAA-3’

Sbe-2a-s2：5’-ATGATCAAGCCTGCTGAATCC-3’

Simultaneously, adopting β-Actin is the internal standard gene of sxemiquantitative PCR, and its primer sequence is as follows:

β-Actin-F：5’-CAGCAACTGGGATGATATGG-3’

β-Actin-R：5’-ATTTCGCTTTCAGCAGTGGT-3’

The result as shown in Figure 7, wherein, swimming lane 1-6: positive T1 is 4-4-1,4-4-2,4-4-3,4-4-8,4-4-11,4-4-19 for changeing the strain of sbeIR wheat; Swimming lane 7: the wild-type wheat, as can be seen, positive T1 illustrates that for changeing the product that sbeIR wheat strain system and wild-type all obtain 1.0KB the SbeIIa gene all has expression, but the abundance difference.

Analyze the electrophoretic glue figure of sxemiquantitative with AlphaImager 5500 image analysis software, extract confidential reference items β-Actin among the software analysis result and the Area value of goal gene SbeIIa respectively, both compare then, obtain the relative expression quantity of SbeIIa gene between transgenic line and the negative control (the wild-type wheat protects rich 104), the experiment triplicate, results averaged, the result is as shown in Figure 8: 4-4-1,4-4-2,4-4-3,4-4-8,4-4-11,4-4-19 is respectively positive T1 for changeing the sbeIR wheat, CK protects rich 104 (its SbeIIa gene expression amount is decided to be 1.00) for the wild-type wheat, as can be seen

The SbeIIa gene relative expression quantity of 4-4-1 is 0.97 ± 0.105;

The SbeIIa gene relative expression quantity of 4-4-2 is 1.02 ± 0.298;

The SbeIIa gene relative expression quantity of 4-4-3 is 1.11 ± 0.581;

The SbeIIa gene relative expression quantity of 4-4-8 is 0.83 ± 0.347;

The SbeIIa gene relative expression quantity of 4-4-11 is 0.47 ± 0.313;

The SbeIIa gene relative expression quantity of 4-4-19 is 0.77 ± 0.383;

As can be seen from Figure, the little contrast non-transgenic (the wild-type wheat protects rich 104) that is higher than of the SbeIIa genetic expression of 4-4-2,4-4-3, the SbeIIa of 4-4-1 expresses and is lower than contrast slightly, the SbeIIa genetic expression of 4-4-8,4-4-11,4-4-19 is starkly lower than contrast non-transgenic plant, can illustrate that RNAi expresses member the SbeIIa gene of transfer-gen plant 4-4-1,4-4-8,4-4-11,4-4-19 is had in various degree restraining effect.

From positive T ₁Gather in the crops seed on the generation commentaries on classics sbeIR wheat, sowing continues to go down to posterity, up to obtaining T ₅In generation, changeed sbeIR wheat strain system.Adopt aforesaid method to identify, obtain 224 positive T ₅In generation, changeed sbeIR wheat strain system.

Two, change the Function Identification of sbeIR wheat

1, the principle of amylose content determination

Starch and iodine form the iodine-starch mixture, and have special color reaction.Amylopectin and iodine form the red-brown mixture, and amylopectin and iodine form the mazarine mixture.Under the constant condition of starch total amount, these two kinds of starch dispersion liquids are pressed different ratios to be mixed, under certain wavelength and acidity with the iodine effect, generation by purplish red to dark blue a series of colors, represent its different direct-connected starch and amylopection content ratio, linear according to absorbancy and amylose content, the content of mensuration amylose starch.

2, instrument, equipment

1) pulverizer (experiment Cyclone mill).

2) DPCZ-II type amylose starch determinator.

3) analytical balance: (sensibility reciprocal is 0.0001g).

4) glassware: the 100mL volumetric flask, glass stick, suction pipe, transfer pipet (10mL, 5mL, 1mL).

3, reagent preparation

1) sodium hydroxide (GB629-77) analytical pure, accurately the NaOH solution of the 1mol/L that demarcates;

Take by weighing the 4g sodium hydrate solid, dissolve in the distilled water of 100mL, put into Plastic Bottle.

2) acetic acid (GB676-78) analytical pure, accurately the HAC solution of the 1mol/L that demarcates;

With pure acetic acid 6mL, with distilled water diluting to 100mL.

3) iodine storing solution and iodine reagent:

Title 2g iodine (analytical pure) and 20g potassiumiodide (analytical pure) are with dissolved in distilled water and be diluted to 100mL, are a storing solution.Get 10mL iodine storing solution and be diluted to 100mL, be iodine reagent.

4) sodium hydroxide analytical pure, accurately the NaOH solution of the 0.09mol/L that demarcates;

Get the NaOH solution of 9mL 1mol/L, to 100mL, be the NaOH solution of 0.09mol/L with distilled water diluting.

5) amylose starch standardized solution

Take by weighing weight 0.1000g purity and be 95% potato amylose starch (Harbin Ministry of Agriculture cereal and goods quality inspection center, Potato Amylose-1.0), put into the 100mL volumetric flask, add the wetting sample of 1mL dehydrated alcohol, the sodium hydroxide solution that adds 9mL 1mol/L again heats 10min in boiling water bath, after the cooling, water is settled to 100mL rapidly.

6) rice amylopectin standardized solution

Take by weighing weight 0.1000g purity and be 83.4% rice amylopectin (Harbin Ministry of Agriculture cereal and goods quality inspection center, Rice Amylopectin-1.0), put into the 100mL volumetric flask, add the wetting sample of 1mL dehydrated alcohol, the sodium hydroxide solution that adds 9mL 1mol/L again heats 10min in boiling water bath, after the cooling, water is settled to 100mL rapidly.

7) testing sample solution

Testing sample takes by weighing testing sample 0.1000g after sieving with 80 mesh sieves, puts into the 100mL volumetric flask, add the moistening sample of 1mL dehydrated alcohol, the sodium hydroxide solution that adds 9mL 1mol/L again heats 10min in boiling water bath, after the cooling, water is settled to 100mL rapidly.

4, determination step

1) gets the positive T of following numbering respectively ₅In generation, changeed 100 seeds of sbeIR wheat strain system, and the adding suitable quantity of water is moistening, seals in valve bag and deposits about 24 hours: B143, B187, B085, B108, B179, B186, B024, B114, B109, B134, B315, B088, B062, B189.With wild-type wheat (CK) is contrast.

2) use the Cyclone mill flour milling, each sample mill 3 times is crossed 80 mesh sieves, and gained flour is packed in the new valve bag, and is standby, promptly obtains thick starch sample.

3) baseline calibration

Get the volumetric flask of a 100ml, add volumetric flask that the NaOH solution 5ml of 0.09mol/L puts into 100ml, add acetic acid, the 1mL iodine reagent of 50mL distilled water, 1mL 1mol/L then successively, be settled to the 10min that develops the color behind the 100mL with distilled water as blank.Baseline calibration solution as DPCZ-2 type amylose starch determinator.

4) mixing working curve draws

Baseline calibration finishes, and draws and mixes working curve (standard working curve)

Get 6 100mL volumetric flasks, add 1mg/mL potato amylose starch standardized solution 0,0.25,0.50,1.00,1.50,2.00mL respectively, add rice amylopectin standardized solution 5,4.75,4.50,4.00,3.50,3.00mL more successively, total amount is 5mL.Other gets the volumetric flask of 1 100mL, and the NaOH solution 5mL that adds 0.09mol/L does blank.The acetic acid and the 1mL iodine reagent that in each bottle, add about 50mL water, 1mL 1mol/L then successively.10min develops the color behind the water constant volume.

Measure with DPCZ-2 type amylose starch determinator, draw and provide standard working curve by system software.

5) testing sample is measured

(1) sample dispersion: take by weighing the thick starch sample of 0.1000g in the 100mL volumetric flask, add the 1mL dehydrated alcohol, abundant wetting sample, the sodium hydroxide solution that adds 9mL 1mol/L again disperses 10min in boiling water bath, cooling rapidly, water constant volume.

(2) degreasing: get the 20mL dispersion liquid and possess in the scale test tube in 50mL, add the 7-10mL sherwood oil, intermittently shake 10min, static 15min with the suction pipe suction that is connected on the water pump, inhales and removes the top petroleum ether layer after the layering, repeats above operation 2-3 time.

(3) get testing sample solution 5mL, put into the 100mL volumetric flask.The acetic acid, the 1mL iodine reagent that add 50mL distilled water, 1mL 1mol/L successively.Be settled to the 10min that develops the color behind the 100mL with distilled water.Measure its amylose content with DPCZ-2 type amylose starch determinator.

Result such as table 1 and shown in Figure 9, among Fig. 9, CK is that the wild-type wheat " protects rich 104 ", all the other are the positive T that is numbered B143, B187, B085, B108, B179, B186, B024, B114, B109, B134, B315, B088, B062, B189, B024 ₅In generation, changeed the sbeIR wheat.

It is the percentage composition of amylose starch in the flour that table 1 changes the sbeIR strain

The check and analysis of table 2 significance

F _0.05(14，28)＝2.31 F _0.01(14，28)＝3.34

The PLSD method, S=sqrt (2MSe/r)=sqrt (0.114869333)=0.3389

PLSD _0.05＝2.048×0.3389＝0.694

PLSD _0.01＝2.763×0.3389＝0.936

From The above results as can be seen, B109 significantly improves than wild-type, and B134, B315, B088, B062, these 5 strain systems of B189 significantly improve than the amylose content utmost point of wild-type (contrast).Illustrate that this sbeIR member can improve the content of amylose starch in the wheat grain.

Claims (10)

1. RNA molecule, its nucleotides sequence is classified the sequence 4 in the sequence table as.

2. dna molecular of expressing the described RNA of claim 1, its nucleotides sequence is classified the sequence 5 in the sequence table as.

3. the recombinant expression vector, transgenic cell line, reorganization bacterium or the expression cassette that contain the described dna molecular of claim 2.

4. recombinant expression vector according to claim 3 is characterized in that: described recombinant expression vector is for inserting the carrier that obtains between the multiple clone site of pBAC47P carrier with the described dna molecular of claim 2.

5. the amplification described dna molecular total length of claim 2 and any segmental primer thereof are right.

6. described RNA molecule of claim 1 or the described dna molecular of claim 2 application in the quality of improvement wheat starch.

7. application according to claim 6 is characterized in that: the quality of described wheat starch embodies by the wheat grain amylose content.

8. a method of cultivating the transgenic plant of high amylose content is with the forfeiture of the SBEIIa gene function in the purpose plant, obtains transgenic plant, and the amylose content in the seed of described transgenic plant is higher than described purpose plant.

9. method according to claim 8 is characterized in that: described SBEIIa gene function in purpose plant forfeiture will be realized by will the described dna molecular of claim 2 importing in the purpose plant.

10. it is characterized in that according to Claim 8 or 9 described methods: the described dna molecular of claim 2 imports by claim 3 or 4 described recombinant expression vectors;

Described purpose plant is monocotyledons or dicotyledons;

The nucleotides sequence of described SBEIIa gene is classified the sequence 1 in the sequence table as.

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