CN102747100A - New method for formulation of full waxy starch wheat germplasm - Google Patents

Abstract

The invention discloses a method for cultivation of full waxy wheat. The method for cultivation of full waxy wheat provided in the invention includes the following step of: inhibiting the expression of GBSS (granule-bound starch synthase) I gene in target wheat to obtain the full waxy wheat. The inhibition of expression of the GBSS I gene in the target wheat is realized by transferring a DNA fragment as shown in formula "SEQforward-X-SEQbackward" into the target wheat. Specifically, the SEQforward is the top 288 nucleotides in sequence 1 of a sequence table; the SEQbackward is in reverse complement with the SEQforward; the X is a spacer sequence between the SEQforward and the SEQbackward, and in sequences, the X is not complementary with and the SEQforward and the SEQbackward. Experiments prove that the wheat GBSS I gene can be silenced through a transgene silencing technology (RNAi) to obtain a GBSS I gene expression-inhibiting full waxy wheat new germplasm, and the transgenic material has excellent comprehensive agronomic characters, thus being able to be directly used for selective breeding of wheat low-amylose new strains (varieties) or be used as a patent material of breeding.

Description

A kind of novel method of complete glutinous matter starch wheat germplasm initiative

Technical field

The invention belongs to biological technical field; Relate to a kind of method of cultivating full Waxy wheat; Be particularly related to a kind ofly,, thereby obtain the method for full Waxy wheat new germ plasm wheat grain mating type starch synthase gene (GBSS I) silence through transgene silencing technology (RNAi).

Background technology

The genetic improvement of wheat starch quality is called as " biotechnology that starch is become gold " abroad, and Waxy wheat (waxy wheat) is a research focus wherein.

Do not contain amylose starch or content few (usually less than 1%) in the full Waxy wheat seed starch; The starch viscosity characteristic and the common wheat of its flour have very big difference; It is a kind of brand-new wheat lines; Can be widely used in industries such as food-processing, Speciality Foods exploitation and relevant with starch processes raw material, papermaking, weaving, medicine, have very big development and application values.In foodstuffs industry; Waxy wheat is as a kind of novel raw material; Characteristics such as have that the retrogradation resistance is big, water-absorbent is strong, the dough snappiness is good can replace other glutinous property raw material such as glutinous rice to develop new food, are used with common wheat; The quality of existing food such as noodles, bread, steamed bun can be improved, and the shelf-life of bread and some quickfrozen foods can be prolonged.On industrial application, can replace other waxy starch and do industrial raw material, as producing medicine dressing, industrial alite paste etc.In liquor industry, Waxy wheat possibly replace glutinous rice, practice thrift greatly cost, brew novel sapor the wine article, create new making method etc.Utilize dissimilar disappearance material hybrid methods, corn and wheat hybridizing generation haplomethod and induce glutinous property mutantion line method; Cultivate protein-high strong muscle type Waxy wheat or high-quality and the low part waxy wheat kind of amylose content, should become the important measures of modified starch quality.

In the crop endosperm, storage starch comprises two types of amylose starch and pulullan, and what of crop amylose content are determining the size of its range of application to a great extent, and amylose content is the important indicator of measurement crop quality.Particle mating type amylosynthease I is an amylose starch synthetic key enzyme in plant; GBSS I is controlling the synthetic of amylose starch in the gramineous crop grain endosperm; (also claim the Waxy gene by the GBSSI gene; Be abbreviated as Wx) encode, in the very low glutinous barley endosperm of amylose content, lack GBSSI with normal function.Glutinous matter or part waxy wheat starch can improve the noodles processing quality; And the waxy wheat of China all is to plant three deletion segment polymerizations of GBSS I seed selection through farmers' to form, and is relatively poor and can not directly be used for wheaten food processing and be difficult to wide range of commercial and use because of economical character.

Summary of the invention

The purpose of this invention is to provide a kind of method of cultivating full Waxy wheat.

The method of the full Waxy wheat kind of cultivation provided by the present invention comprises the steps: to suppress GBSS I expression of gene in the purpose wheat, obtains full Waxy wheat.

In the present invention, GBSS I expression of gene realizes through changing in the purpose wheat as shown in the formula the dna fragmentation shown in (1) in the said inhibition purpose wheat:

SEQ _Forward-X-SEQ _Oppositely(1)

Said SEQ _ForwardBe preceding 288 Nucleotide of sequence 1 in the sequence table;

Said SEQ _OppositelySequence and said SEQ _ForwardThe sequence reverse complemental;

Said X is said SEQ _ForwardWith said SEQ _OppositelyBetween intervening sequence, on sequence, said X and said SEQ _ForwardAnd said SEQ _OppositelyAll not complementary.

The aminoacid sequence of said GBSS I genes encoding is a sequence 2 in the sequence table, and its albumen is wheat grain mating type amylosynthease I.Sequence 2 is made up of 604 amino acid.

The said complete glutinous content that does not contain amylose starch or amylose starch in the wheat grain starch very low (the quality percentage composition is lower than 1%) that is meant.

Another object of the present invention provides a kind of method of wheat that amylose content reduces of cultivating.

The method of wheat that cultivation amylose content provided by the present invention reduces comprises the steps: to suppress GBSS I expression of gene in the purpose wheat, obtains comparing with said purpose wheat the wheat of amylose content reduction.

In the present invention, GBSS I expression of gene realizes through changing in the purpose wheat as shown in the formula the dna fragmentation shown in (1) in the said inhibition purpose wheat:

SEQ _Forward-X-SEQ _Oppositely(1)

Said SEQ _ForwardBe preceding 288 Nucleotide of sequence 1 in the sequence table;

Said SEQ _OppositelySequence and said SEQ _ForwardThe sequence reverse complemental;

Said X is said SEQ _ForwardWith said SEQ _OppositelyBetween intervening sequence, on sequence, said X and said SEQ _ForwardAnd said SEQ _OppositelyAll not complementary.

The aminoacid sequence of said GBSS I genes encoding is a sequence 2 in the sequence table, and its albumen is wheat grain mating type amylosynthease I.Sequence 2 is made up of 604 amino acid.

In the present invention, the nucleotide sequence of the dna fragmentation shown in all above-mentioned formulas (1) all is specially sequence 1 in the sequence table.

Sequence 1 is made up of 1054 Nucleotide.Wherein, preceding 288 is a segmental forward sequence (SEQ in the corresponding above-mentioned formula (1) of the gene (GBSSI gene) of wheat grain mating type amylosynthease Id _Forward), the 289-766 position is paddy rice intron nucleotide sequence (X in the corresponding above-mentioned formula (1)), back 288 is segmental reverse sequence of wheat grain mating type amylosynthease GBSSI gene (SEQ in the corresponding above-mentioned formula (1) _Oppositely).

In one embodiment of the invention, the dna fragmentation shown in the said formula (1) specifically is that form through the rnai expression carrier changes in the purpose wheat; The promotor that the dna fragmentation shown in the said formula of startup (1) is transcribed on the said rnai expression carrier is the CaMV35S promotor.More concrete, said rnai expression carrier is for inserting the RNA interference sequence (sequence 1) of said wheat grain mating type amylosynthease I (GBSSI), the recombinant plasmid that obtains at the MCS place of pCAMBIA3301 carrier; Said MCS is specially Bg1II and EcoR I.

In one embodiment of the invention, above-mentioned all purpose wheats all are specially wheat breed section farming 199

Utilize the said full Waxy wheat kind of aforesaid method acquisition or the wheat breed of said amylose content reduction also belonging to protection scope of the present invention as the application among the wheat breeding parent.

Dna fragmentation as shown in the formula shown in (1) also belongs to protection scope of the present invention:

SEQ _Forward-X-SEQ _Oppositely(1)

Said SEQ _ForwardBe the 1-288 position Nucleotide of sequence 1 in the sequence table;

Said SEQ _OppositelySequence and said SEQ _ForwardThe sequence reverse complemental;

Said X is said SEQ _ForwardWith said SEQ _OppositelyBetween intervening sequence, on sequence, said X and said SEQ _ForwardAnd said SEQ _OppositelyAll not complementary.

In the present invention, the nucleotide sequence of said dna fragmentation is specially sequence table sequence 1.

The recombinant vectors, reorganization bacterium, expression cassette or the transgenic cell line that contain the dna fragmentation shown in the said formula (1) also belong to protection scope of the present invention.

Said recombinant vectors both can be recombinant expression vector, also can be recombinant cloning vector.In one embodiment of the invention; The promotor that the said RNA interference sequence of startup is transcribed in the said recombinant expression vector is the CaMV35S promotor; More concrete; Said recombinant expression vector goes out to insert the dna fragmentation shown in the said formula (1) (sequence 1), the recombinant plasmid that obtains for the MCS at the pCAMBIA3301 carrier; Said MCS is specially Bg1II and EcoR I.

The experiment proof; Through transgene silencing technology (RNAi); Gene (GBSS I gene) silence with wheat grain mating type amylosynthease I; To suppress the full Waxy wheat new germ plasm of GBSS I genetic expression, the comprehensive agronomy proterties of transgenic line is good, can directly be used for seed selection wheat low amylose starch new lines (kind) or as the breeding parent material.

Description of drawings

Fig. 1 is T ₀PCR detected result for transgenic wheat.Wherein, swimming lane 1 is a blank; Swimming lane 2-11 is 10 T ₀For the transgenic wheat plant; Swimming lane 12 positive contrasts; Swimming lane 13 negative contrasts; Swimming lane M is the Marker molecular weight standard.

Fig. 2 is T ₀I for the transgenic wheat seed ₂-KI coloration result.Wherein, a left side is the seed coloration result of wild-type wheat breed section farming 199; The right side is T ₀For transgenic wheat Wax-1 seed coloration result.

Fig. 3 is T ₀SDS-PAGE electrophoresis result for the transgenic wheat seed.Wherein, swimming lane 1 is T with swimming lane 2 ₀For farming Wax-1 of transgenic wheat section and Wax-2; Swimming lane 3 is wild-type wheat breed section farming 199.

Fig. 4 detects T for RT-PCR ₃Result for GBSS I genetic expression in the transgenic wheat seed.Wherein, swimming lane 1 is wild-type wheat breed section farming 199; Swimming lane 2 is T ₃For transgenic wheat Wax-1-3.

Fig. 5 is T ₅For the farming 199/pCAMBIA3301-GBSSI of transgenic wheat section seed starch gelatinization plots changes figure.Wherein, A is wild-type wheat breed section farming 199; B is T ₅For the farming 199/pCAMBIA3301-GBSSI of transgenic wheat section.

Embodiment

Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.

Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.

The pCAMBIA3301 carrier is available from Beijing DingGuo ChangSheng Biology Technology Co., Ltd's (article No.: MCV039).

The pTCK303 carrier: effluent north professor Sun Ying of normal university is so kind as to give.Reference: Zhang Rui, Hu Wenquan, Xie Wanqin, Li Cuifeng. Chinese cabbage changes the preliminary foundation of the reticent system of RNAi of lipoprotein gene CaMBP10. Chinese agronomy circular .2011,27 (10): 168-172.

The pAHC25 carrier is provided by kingly way doctor Wen of Inst. of Genetics and Development Biology, CAS.Reference: Rita Abranches, Ana P.Santos, Eva Wegel; Sarah Williams; Alexandra Castilho, Paul Christou, Peter Shaw; Eva Stoger.Widely separated multiple transgene integration sites in wheat chromosomes are brought together at interphase.The Plant Journal (2000) 24 (6), 713-723.

Wheat breed section farming 199: reference: Zhao Hui, Zhang Wei, Wang Jing; Discipline army; Wang Zhiguo, Li Junming. New Winter Wheat Variety " section's farming 199 " stable high yield signature analysis. Chinese Ecological Agriculture journal, 2011; 19 (5): in the 1220-1228. wheat breed section farming 199 amylose starch and amylopection content about 17%, be the common wheat kind.

The detection of the acquisition of embodiment 1, transgenic wheat and Wx protein expression situation thereof

One, the structure of RNAi expression vector pCAMBIA3301-GBSS

At first the pCAMBIA3301 carrier is carried out following transformation,, cut product with 1% agarose gel electrophoresis purifying and recovery enzyme with Kpn I and Spe I double digestion pTCK303 carrier.The paddy rice intron sequences that one section size that enzyme is downcut is 478bp is connected to pMD19-T carrier (Takara (Dalian) company; D102A) on; With the recombinant plasmid called after pMD19-T-intron that obtains; It is converted into DH5 α competent cell, and the picking positive colony send company to carry out sequencing.After the result that waits to check order is correct,, the paddy rice intron sequences is connected the CaMV35S promotor of pCAMBIA3301 carrier and the MCS between the NOS terminator with Kpn I and Spe I double digestion pMD19-T-intron and pCAMBIA3301 carrier.Specific as follows:

With GBSS I cDNA (GenBank:Y16340.1) sequence is template; And according to its sequences Design 2 to (forward with reverse) special primer; Introduce restriction enzyme site respectively; Utilize the primer (primer 1 and primer 2) of this gene forward sequence of a pair of amplification; And the primer of this gene reverse sequence of a pair of amplification (primer 3 with primer 4) respectively amplification obtain the forward fragment that two ends have restriction enzyme site BglII and Xba I, and two ends have the reverse fragment of restriction enzyme site EcoR I and Kpn I, and are connected with pMD19-T simple carrier (Takara company) respectively.Then utilize restriction enzyme BglII and Xba I double digestion to be connected the forward fragment on the pMD19-T simple carrier, it is connected into through among the expression vector pCAMBIA3301 that is connected with the CaMV35S promoter fragment that transforms, form recombinant plasmid 1.Utilize restriction enzyme EcoR I and Kpn I double digestion to be connected the reverse fragment on the pMD19-T carrier again, its above-mentioned recombinant plasmid 1 with the same double digestion of process is linked to each other, form recombinant plasmid 2.The both sides that forward fragment and reverse fragment are inserted intron on the carrier are respectively separated to keep the stability of carrier by the intron sequences on one section carrier between forward fragment and the reverse fragment; This system transcribes the dsRNA that produces band hairpin structure (hairpin) in vegetable cell, cause RNAi, thereby suppresses the expression of goal gene.

Primer 1:5 '-AT AGATCTCTCGTACCAGGGCCGCTTCT-3 ' (the underscore place is the recognition sequence of Bg1II, and sequence thereafter is preceding 20 of sequence 1)

Primer 2: 5 '-AG TCTAGAGGGTCCCACTCGCTAACATCC-3 ' (the underscore place is the recognition sequence of Xba I, and sequence thereafter is the reverse complementary sequence of the 268-288 position of sequence 1)

Primer 3:5 '-G GAATTCGGTTACCCTCGTACCAGGGCCGCTTCT-3 ' (the underscore place is the recognition sequence of EcoR I, and the 15-34 position of this sequence is back 20 reverse complementary sequence of sequence 1)

Primer 4:5 '-TA GGTACCGGGTCCCACTCGCTAACATCC-3 ' (the underscore place is the recognition sequence of Kpn I, and sequence thereafter is the 767-787 position of sequence 1)

Recombinant plasmid 2 is checked order the recombinant plasmid 2 called after pCAMBIA3301-GBSS I (being the rnai expression carrier of wheat grain mating type amylosynthease GBSSI) of sequence 1 in the insertion sequence table between the Bg1II that will pass through checks order is illustrated in the pCAMBIA3301 carrier and the EcoR I/restriction enzyme site.Sequence 1 is the RNA interference sequence among the RNAi expression vector pCAMBIA3301-GBSS, is made up of 1054 Nucleotide.Wherein, Preceding 288 is a segmental forward sequence of wheat grain mating type amylosynthease GBSSI gene; The 289-766 position is a paddy rice intron nucleotide sequence, and back 288 is a segmental reverse sequence of wheat grain mating type amylosynthease GBSSI gene.In the pCAMBIA3301-GBSS I, starting the promotor that said RNA interference sequence (sequence 1) transcribes is the CaMV35S promotor.

Two, the acquisition of transgenic wheat

The RNAi expression vector pCAMBIA3301-GBSS of the wheat grain mating type amylosynthease I GBSS I that step 1 is made up with the method for particle gun bombardment and the rataria of the pAHC25 carrier cotransformation wheat breed section farming 199 that carries the bar gene.Be provided with simultaneously and change the pCAMBIA3301 empty carrier over to for contrasting.Owing to contain the bar gene on the pAHC25 carrier, can be used as the selection markers of the two third ammonia phosphorus (PPT) of antiweed.

Wheat immature embryo after transforming is placed the enterprising row filter of screening culture medium (the 1/2MS substratum adds PPT, and making the PPT final concentration is 4mg/L) of the selective agent PPT that contains 4mg/L, obtain the transgenic positive plant of antiweed.

Antiweed transgenic positive plant to above-mentioned acquisition carries out the PCR Molecular Identification; Genomic dna with the transgenic positive plant of the antiweed of above-mentioned acquisition is a template, carries out pcr amplification (one section sequence on the amplification vector between terminator and reverse sequence) with upstream primer and downstream primer.Following three contrasts are set simultaneously: blank-replace template with the pCAMBIA3301 carrier; Positive control-replace template with the pCAMBIA3301-GBSS carrier; Negative control-with the genomic dna of not genetically modified section farming 199 is a template.

Upstream primer: 5 '-AGTAACATAGATGACACCGCGC-3 '

Downstream primer: 5 '-GGTCCCACTCGCTAACATCCA-3 ' (the 768-788 position of sequence 1)

The result is as shown in Figure 1, and obtaining size through pcr amplification is the positive T that changes wheat grain mating type amylosynthease GBSS I rnai expression carrier pCAMBIA3301-GBSS over to for the plant of 535bp purpose band ₀For transfer-gen plant (Fig. 1 swimming lane 2-11), with its called after wax-1 to wax-10 successively.For the transgenic wheat that changes the pCAMBIA3301 empty carrier over to, amplification does not have the purpose band of target sequence.

Three, the glutinous property evaluation of transgenic wheat

This research will detect from the glutinous property of following two aspects to transgenic wheat: one of which, carry out I to wheat seed ₂-KI dyeing, thereby the glutinous property and the non-glutinous property of judgement transgenic wheat.Amylose content is low more, and the glutinous property of wheat is strong more.Its two, the waxy proteins of wheat is carried out the SDS-PAGE electrophoresis, thereby judges the proteic expression of Wx in the wheat.The proteic expression amount of Wx is low more, and the glutinous property of wheat is strong more.

1, I ₂The proteic expression of Wax in the transgenic wheat seed is identified in-KI dyeing

T with the step 2 acquisition ₀Seed for transgenic wheat is an experiment material, carries out I ₂The moity of its seed starch of-KI staining analysis (amylose starch or pulullan).Concrete operations are following: seed is cut embryo end far away with cutter, and remainder is used 0.2% I ₂～2% KI reagent dyeing is behind the colour developing 5min, according to " amylose starch is met iodine and shown mazarine, and pulullan is met iodine and shown reddish-brown " this standard observations.Simultaneously with farming 199 of wild-type wheat breed section and T ₀In generation, change the seed of transgenic wheat of pCAMBIA3301 empty carrier over to as contrast.All seeds of each plant experimentize.

The result is as shown in Figure 2, T ₀Seed section for transgenic wheat Wax-1 is reddish-brown, and wild-type wheat breed section farming 199 is mazarine with the seed section that changes the transgenic wheat of pCAMBIA3301 empty carrier over to.This result shows, T ₀Be substantially free of amylose starch for transgenic wheat Wax-1 seed, show stronger glutinous property.

2, Wax protein expression situation in the SDS-PAGE electrophoresis detection transgenic wheat

T with the step 2 acquisition ₀All seeds for transgenic wheat Wax-1 and Wax-2 are experiment material, extract waxy proteins, carry out SDS-PAGE electrophoresis detection Wx protein expression situation then.Concrete operations are following:

Cut the embryo end (reservation) of sophisticated wheat seed, remaining half is for use in managing with the broken 1.5mL EP that places of flat nose pliers folder.In the EP pipe, add the dual steaming of 0.5mL and stay water, put into 4 ℃ of following lixiviate >=5h of refrigerator, remove kind of a skin, 4 ℃ of centrifugal 5min of 10000r/min abandon supernatant.Add 0.5mL bufferA1 (55mmol/L Tris-HC1, pH6.8,2.3% (w/v) SDS, 5% (v/v) 2 mercapto ethanol, 10% (v/v) glycerine), 4 ℃ of centrifugal 5min of 10000r/min abandon supernatant, repeat 3 times.Add 0.5mL ddH ₂O, mixing, 4 ℃ of centrifugal 5min of 10000r/min abandon supernatant, repeat 2 times.Add 0.5mL acetone, mixing, 4 ℃ of centrifugal 5min of 10000r/min abandon supernatant, repeat 2 times.Throw out is vacuum-drying 5min in Vacuumdrier, the starch small grain that obtains purifying.Add 100 μ L extracting solution bufferA2 (55mmol/L Tris-HCl, pH6.8,2.3% (w/v) SDS by every 10mg dry starch grain; 5% (v/v) 2 mercapto ethanol, 10% (v/v) glycerine, 0.005% (v/v) bromjophenol blue); Mixing, boiling water bath 5min, 4 ℃ of centrifugal 5min of 10000r/min; Supernatant is used for point sample, carries out the proteic expression of Wx in the SDS-PAGE electrophoretic analysis transgenic wheat.While is with the seed and the T of wild-type wheat breed section farming 199 ₀The transgenic wheat that generation changes the pCAMBIA3301 empty carrier over to is as contrast.The experiment triplicate.

The result is as shown in Figure 3, T ₀Three sites for Wx in the seed of transgenic wheat Wax-1 and Wax-2 all do not have protein band, and have detected proteic three subunits of Wx simultaneously as the wild-type wheat breed section farming 199 of contrast, i.e. Wx-A1, Wx-B1 and Wx-D1.The transgenic wheat detected result that changes the pCAMBIA3301 empty carrier over to is identical with wild-type wheat breed section agricultural 199.This shows, T ₀Has stronger glutinous property (glutinous entirely) for transgenic Wax-1 and Wax-2.

Four, the detection of transgenic wheat genetic stability

T with the step 2 acquisition ₀Carry out continuous self propagated for transgenic wheat Wax-1, obtain T ₃For transgenic wheat Wax-1-3.Choosing the seed that the Wax-1-3 transfer-gen plant bloomed 15 days is material, carries out sxemiquantitative RT-PCR, the expression of research GBSSI gene in two mutants, with Actin as internal control gene.The primer of GBSSI gene of being used to increase is 5 '-CCGACGAACACGAGGTTCATG-3 ' (reverse complementary sequence of the 383-403 position of GenBank:Y16340.1) and 5 '-ACTCGCTAACATCCATGCCG-3 ' (reverse complementary sequence of the 1196-1215 position of GenBank:Y16340.1; The primer of Actin gene of being used to increase is 5 '-GTTCCAATCTATGAGGGATACACGC-3 ' and 5 '-GAACCTCCACTGAGAACAACATTACC-3 '.While is with the seed and the T of wild-type wheat breed section farming 199 ₃The transgenic wheat that generation changes the pCAMBIA3301 empty carrier over to is as contrast.Each strain system chooses 3 strains and experimentizes.The experiment triplicate.

The result is as shown in Figure 4, at T ₃Under the situation for the expression amount basically identical of the internal control gene Actin of transgenic wheat Wax-1-3 and wild-type section farming 199, GBSSI gene normal expression in the farming 199 of wild-type section, and T ₃Almost do not express for GBSSI gene among the transgenic wheat Wax-1-3.For T ₃In generation, change the transgenic wheat of pCAMBIA3301 empty carrier over to, and its detected result is with wild-type section farming 199.Above result shows that the GBSSI gene do not express on transcriptional level, can be in transgenic wheat genetic stability.Simultaneously with one of them T ₃For the transgenic wheat strain is called after Wx-1-3-1, is used for the detection of following starch quality characteristic.

Embodiment 2, transgenic wheat offspring's starch quality Characteristics Detection

One, transgenic wheat offspring's seed starch and determining the protein quantity

T with embodiment 1 acquisition ₃Seed for transgenic wheat Wax-1-3-1 is an experiment material, carries out the starch quality Characteristics Detection.Concrete operations are following: seed starch content is pressed GB/T1568521995 polarimeter method and is measured.Amylose content adopts dual-wavelength spectrophotometry to measure in the wheat grain, and the predominant wavelength of amylose content determination is 580nm, and reference wavelength is 500nm.The seed total nitrogen content originates from moving azotometer (nitrogen determination) with Switzerland to be measured, and the seed total nitrogen content multiply by 5.7 and is grain protein content.While is with the seed and the T of wild-type section farming 199 ₃The transgenic wheat that generation changes the pCAMBIA3301 empty carrier over to is as contrast.Each plant selects 20 seeds to experimentize.The experiment triplicate, results averaged.

The result is as shown in table 1, and the amylose content of wild-type wheat breed section farming 199 is 17.22%, T ₃Be merely 0.22%, significant difference for amylose content in the seed of transgenic wheat Wx-3-1; But T ₃For total starch content and crude protein content in the seed of transgenic wheat Wx-3-1 all with wild-type wheat breed section farming 199 no significant differences.

Table 1 transgenic wheat offspring and wild-type wheat grain starch and protein content

Type	Total starch content %	Protein contnt %	Amylose content %
				Wx-1-3-1	64.21±2.57	14.68±0.82	0.22±0.092
Section's farming 199	65.89±2.39	15.35±0.95	17.22±0.076

Annotate: content described in the table is the quality percentage composition.

Two, the mensuration of transgenic wheat offspring's seed starch gelatinization characteristic relation conefficient

T with embodiment 1 acquisition ₃Carry out self propagated twice for transgenic wheat Wax-1-3-1, obtain T ₅For transgenic wheat (the farming 199/pCAMBIA3301-GBSS of called after section), gather in the crops its seed as experiment material, with the seed and the T of wild-type wheat breed section farming 199 ₅The transgenic wheat that generation changes the pCAMBIA3301 empty carrier over to carries out the mensuration of starch pasting characteristic relation conefficient as contrast.Concrete operations are following:

Measure 25 gram zero(ppm) water earlier and put into container, take by weighing 3 gram flour again, stir fast several down after, with the quick viscosity apparatus mensuration of the RVA-4500 of Sweden perten manufactured starch pasting characteristic.Mode determination selection standard method 1 and standard method of analysis 1.Measure 50 ℃ of starting temperatures, 960r/min mixing 10s, finding speed are 160r/min; The gelatinization stage is warming up to 95 ℃, 4min 32s consuming time, 95 ℃ of constant temperature 3min30s then from 50 ℃; Be cooled to 50 ℃ of 3min 48s consuming time from 95 ℃ subsequently, 50 ℃ of constant temperature 2min, whole test amounts to 13min.Each sample repeats 3 times, results averaged.

The result shown in Fig. 5 and table 2, T ₅Have higher peak viscosity and fall value for transgenic wheat section farming 199/pCAMBIA3301-GBSS seed starch; And T ₅Compare all to have extremely significantly with the wild-type contrast for low ebb viscosity, final viscosity, the rise value of transgenic wheat section farming 199/pCAMBIA3301-GBSS seed starch and reduce, T is described ₅Early starch retrogradation is slow to begin gelatinization for transgenic wheat section farming 199/pCAMBIA3301-GBSS seed starch; Starch gelatinization temperature slightly raises than the gelatinization point (64.3 ℃) of wild-type contrast simultaneously, but the starch time to peak is than time to peak (6.23min) the reduction 2.90min of wild-type starch.This result shows; The farming 199/pCAMBIA3301-GBSS of transgenic wheat section has improved the starch peak viscosity; Shorten time to peak, made the easy gelatinization of starch, improved glutinous gathering property; Improved the gelatinization characteristic of starch, these results have further embodied the extremely strong glutinous property (glutinous entirely) of the farming 199/pCAMBIA3301-GBSS of transgenic wheat section.

The comparison of table 2 transgenic wheat offspring and wild-type wheat grain starch viscosity relation conefficient

Claims (10)

1. a method of cultivating full Waxy wheat comprises the steps: to suppress GBSS I genetic expression in the purpose wheat, obtains full Waxy wheat.

2. method according to claim 1 is characterized in that: GBSS I expression of gene realizes through changing in the purpose wheat as shown in the formula the dna fragmentation shown in (1) in the said inhibition purpose wheat:

SEQ _Forward-X-SEQ _Oppositely(1)

Said SEQ _ForwardBe the 1-288 position Nucleotide of sequence 1 in the sequence table;

Said SEQ _OppositelySequence and said SEQ _ForwardThe sequence reverse complemental;

Said X is said SEQ _ForwardWith said SEQ _OppositelyBetween intervening sequence, on sequence, said X and said SEQ _ForwardAnd said SEQ _OppositelyAll not complementary.

3. cultivate the method for wheat that amylose content reduces for one kind, comprise the steps: to suppress GBSS I expression of gene in the purpose wheat, obtain comparing the wheat of amylose content reduction with said purpose wheat.

4. method according to claim 3 is characterized in that: GBSS I expression of gene realizes through changing in the purpose wheat as shown in the formula the dna fragmentation shown in (1) in the said inhibition purpose wheat:

SEQ _Forward-X-SEQ _Oppositely(1)

Said SEQ _ForwardBe the 1-288 position Nucleotide of sequence 1 in the sequence table;

Said SEQ _OppositelySequence and said SEQ _ForwardThe sequence reverse complemental;

Said X is said SEQ _ForwardWith said SEQ _OppositelyBetween intervening sequence, on sequence, said X and said SEQ _ForwardAnd said SEQ _OppositelyAll not complementary.

5. according to claim 2 or 4 described methods, it is characterized in that: the nucleotides sequence of the dna fragmentation shown in the said formula (1) is classified sequence 1 in the sequence table as.

6. according to claim 2 or 4 or 5 described methods, it is characterized in that: the dna fragmentation shown in the said formula (1) is that the form through the rnai expression carrier changes in the purpose wheat; The promotor that the dna fragmentation shown in the said formula of startup (1) is transcribed on the said rnai expression carrier is the CaMV35S promotor.

7. utilize wheat that arbitrary said method obtains among the claim 1-6 full Waxy wheat or amylose content reduce as the application among the wheat breeding parent.

8. as shown in the formula the dna fragmentation shown in (1):

SEQ _Forward-X-SEQ _Oppositely(1)

Said SEQ _ForwardBe the 1-288 position Nucleotide of sequence 1 in the sequence table;

Said SEQ _OppositelySequence and said SEQ _ForwardThe sequence reverse complemental;

Said X is said SEQ _ForwardWith said SEQ _OppositelyBetween intervening sequence, on sequence, said X and said SEQ _ForwardAnd said SEQ _OppositelyAll not complementary.

9. dna fragmentation according to claim 8 is characterized in that: the nucleotides sequence of said dna fragmentation is classified sequence table sequence 1 as.

10. the recombinant vectors, reorganization bacterium, expression cassette or the transgenic cell line that contain claim 8 or 9 said dna fragmentations.

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