CN1156951A - Novel plants and processes for obtaining them - Google Patents

Abstract

The invention relates to a transgenic or mutated plant having genomic material which alters the normal starch synthesis pathway within the plant. More specifically, the present invention relates to a plant having a genotype which creates new forms of starch in significant quantity. Particularly, the invention relates to grain having an embryo with a genotype heterozygous for two or more wild type genes (for example, Aa/Bb) and an endosperm having a genotype heterozygous for such genes (for example, AAa/BBb or AAa/bbB or aaA/BBb or aaA/bbB) and the starch produced therefrom.

Description

New plant and preparation method thereof

Invention field

The present invention relates to have genetically modified plants or the mutant plant that changes the genomic material of normal starch route of synthesis in the plant.Or rather, the present invention relates to have the genotypic plant that produces a large amount of novel starchs.Specifically, the present invention relates to have the embryo and the cereal of the endosperm of heterozygous genes type (for example AAa/BBb or AAa/bbB or aaA/Bbb or aaA/bbB) and the starch of production thereof of the heterozygous genes type (for example Aa/Bb) of two or more wild type genes with described gene.

Cereal of the present invention produces by a plant species of having taught another kind of plant pollen, a wherein said plant species is for being the homozygous recessive gene type at least a gene, and another gene is wild type (for example aa/BB), described another kind of plant for another gene at least be the homozygous recessive gene type wherein other gene be wild type (for example AA/bb).

Background of invention

Many plants all produce and storage strach.These plants have the route of synthesis that produces starch.The output of starch is with different variation of type of plant.Cereal is known product starch plant.Described cereal comprises rice, corn, Chinese sorghum, barley, wheat, rye and oat.In addition, the known potato section plant of sweet potato and some fruit (as banana) of comprising also produces starch.

Starch is carbon fixation end-product important in the leaf photosynthesis, is the important storage product in seed and the fruit.From economics point, the starch that is partly produced by the edibles of cereal in wheat, rice and the corn three can provide with calorimetric global about 2/3 food.

Plant amylum can be used in many ways.For example, can be used for culinary art and food processing through extracting.Starch can be stayed in cereal or the plant and use for animal and human's class.Starch can also be used to make the distillation processing of wine, for example, starch can be converted into ethanol.In addition, starch can be converted into high fructose syrup and other industrial component.

In dictionary, starch is defined as the granular solids that its chemical nature is a glycoconjugate, can be used in the products such as adhesive, paste, food, cosmetics, medicine.Say that more generally starch is made up of amylose and amylopectin.Amylose and amylopectin are synthetic in plastid chamber (chloroplast of photosynthetic cells or the amyloplast of non-photosynthetic cells).Different plants produces the amylopectin and the amylose of different proportion.And the different branches pattern of amylopectin and the different chain length of amylose and amylopectin will produce different starch properties.Therefore, the fine structure of amylose and amylopectin is different in the different plants, so that branch's chain and chain length have very big variation, thereby produces the new features that are suitable for different purposes.Up to now, existing four preparations have the approach of special nature starch: (i) utilize the starch that extracts in the kindred plant never; (ii) utilize the starch that from the mutant strain of specified plant, extracts; (iii) utilize through the natural of chemical modification and sudden change starch and (iv) utilize through the natural of physical modification and the starch that suddenlys change.Novel starch under the above-mentioned all situations is all valuable because of the new characteristic that starch type provided.

Mutator gene in the known plants influences the character of starch.Identified the mutator gene relevant with various starch in corn, wherein some are also cloned.The characteristic of apparent (phenotype) of the physics according to corn grain or its starch is to these mutator gene names.(Waxy) that these recessive mutator genes comprise the wax shape (WX), contain sugar (Su) (include but not limited to sugar-1 (Sul), sugar-2 (Su2), sugar-3 (Su3) and sugar-4 (Su4)], (du) of dimness, amylose enriching substance (extender) (ae), horn shape (h) and shrunk (sh) [include but not limited to shrunk-1 (sh-1) and shrunk-2 (sh-2).Some recessive gene mutant wherein produce the homotype enzyme of known enzyme in the starch route of synthesis.The recessive mutation allelomorph of these genes is when being homozygosity or when being expressed with capacity in genetically modified plants in plant, cause the reducing fully or almost completely of specific homotype enzymic activity (hereinafter be defined as with enzyme type activity and reduce fully) of certain enzyme in the route of synthesis.This variation in the starch route of synthesis causes and forms the starch with different qualities.

More known kinds make the dissimilar starch of deposits yields.The difference of starch quality type makes it to be applicable to certain purpose, comprises specific processing method or specific final use.Naturally occurring maize mutant body produces the starch of different fine structures, is applicable to various food and other purpose.Although known mutant produces the starch that has changed, the some of them strain is unsuitable for crop breeding and/or the peasant uses.For example, output is very low, and/or is difficult to processing, and/or is difficult for gelation.

In order to produce different starch, people have cultivated single mutation and two mutant plant.The single mutation plant is isozygotied for a kind of recessive mutation gene.For example, wax shape corn, wax shape rice, waxy barley and wax shape Chinese sorghum have wax shape (WX) gene that isozygoty, sudden change.The genotypic Whilst starch of wax shape has only seldom or does not contain amylose, and another kind is called the starch that amylose is rich in the sudden change generation of amylose enriching substance (ae).Two sudden changes are single plants of two kinds of recessive mutation gene pures (or expressing fully).For example, US4 has described the two sudden changes of Wxf11 in 789,738.Many other novel starchs provide in other relevant patent of starch, have all produced double-mutant or Trimutant (US4 for example, 789,557, US4 in these patents, 790,997, US4,774,328, US4,770,71O, US4,798,735, US4,767,849, US4,801,470, US4,789,738, US4,792,458 and US5,009,911 has described naturally occurring maize mutant body, can produce the starch of the different fine structures that are applicable to different food products).Description the present invention according to described patent application is very beat all, does not need double-mutant or Trimutant because the present invention has produced the starch that changes.

Normal starch is defined as being chemical modification or the starch that produced by the plant with expection gene (wild type) of regulating the starch route of synthesis without the people.In order to be easy to read, the recessive mutation gene that two lowercases (for example aa) expression is isozygotied, the not mutated gene (wild type) that two capitalizations (for example AA) expressions is isozygotied, a capitalization and a lowercase (for example Aa) then represent a sudden change with the non-homozygous gene of a cover of sudden change not.The different gene of different letter representations of identical size; " aa/bb " is double-mutant, and " aa/bB " is for closing mutator gene and heterozygous mutant gene merely in the Plant Genome.For the application, any sequence of three letters of oblique line one side can be exchanged, and does not limit the parent that this gene is provided.For example, AAa/bbB is defined as and is equal to aAA/bBb, AaA/Bbb, AaA/bBb, aAA/Bbb etc.

Although corn plant and embryo thereof are dliploid, corn embryosperm is a triploid.The gene dosage that the genotype of endosperm has two gene dosages of coming from female part heredity and comes from pollen or the heredity of androecy part.Therefore, female if single mutation plant " aa " is used as, and with the male hybridization of not mutated plant " AA ", then the endosperm of this female grain is " aaA ".If Tu Bian plant " AA " and mutant plant " aa " hybridization, so that mutant is not as female, then the endosperm of female grain is " Aaa " because two gene dosages from female and gene dosage from male.Traditional teaching material thinks that mutator gene is recessive, and not mutated gene is a dominance; Therefore, has the normal starch that starch that the plant of the gene dosage of " aaA " or " AAA " or " AAa " produces causes desired amount by endosperm.Yet,, produce endosperm with " aaa " gene dosage as the endosperm of female homozygous mutation plant " aa " and homozygous mutation plant " aa " hybridization as androecy.This endosperm produces the starch with the characteristic that is different from normal starch.Similarly, also show the characteristic that is different from normal starch by endosperm for the starch of the two prominent body generation of " aaa/bbb ".The difference of these starch properties can be used for substituting the starch through chemical modification, or uses or be used for food with food, or as the cereal in the system wine, or in general starch industry, using.

Very clear, the cereal of producing the starch with different physical characteristics needs two kinds of mutant plant hybridization, is the recessive cereal that isozygotys to produce for two kinds of genes.Mutant plant has littler predictability than standard plant.

About from two MHs and/or inbreeding body and some single mutants, producing cereal and extracting starch, there is a problem that often occurs.The amount of the starch that produces is less than the amount of starch that not mutated plant produces usually, and loss is also being arranged aspect the integrality of starch granules size and/or starch granules.Known can produce the starch that changes taken place structure double-mutant in its crop the amount of starch of producing relatively low problem can cause the germinating capacity of seed poor.And the starch yield of seed reduces seemingly inevitable, because sudden change causes the normal starch complex functionality of cell to be damaged.This just needs that a kind of production has that starch structure has taken place that change or character have taken place to change but not obvious minimizing productive rate or reduce the approach of the cereal of starch granules size or integrality.

Summary of the invention

The purpose of this invention is to provide the method that has changed the glycoconjugate content of cereal but do not needed the hybrid plant of double-mutant closed crossing of developing.

The present invention also aims to provide the plant that produces the cereal that has changed starch property.

The present invention also aims to provide the genetically modified plants that produce the cereal that has changed starch property.

The present invention also aims to provide the corn plant that for relevant mutant plant, produces altered starch and greater amount starch.

The present invention also aims to provide new plant, contain the incomplete gene that reduces of at least two homotype enzymic activitys that make certain enzyme in the plant amylum route of synthesis.

The present invention also aims to provide the corn plant of the genotype of endosperm for " AAa/BBb or AAa/bbB or aaA/BBb or aaA/bbB ".

The present invention also aims to provide the plant of generation genotype for the endosperm of " wxwxWX/AeAeae ".

The present invention also aims to provide the starch through changing, it produces by having " AAa/BBb or AAa/bbB or aaA/BBb or aaA/bbB " genotypic plant.

The present invention also aims to provide new purposes by corn plant gained starch of the present invention.

The present invention has covered widely and produced endosperm genotype prevailingly to be the middle mutant of AAa/bbB and to comprise that endosperm is the wax shape, the method for mutant in the middle of some of wax shape (wxwxWX/AEAEae).

The method that production has the cereal that has changed amount of starch comprises that plantation can bloom and serve as female parent's plant.Female parent reduces basically fully at least a specific homotype enzymic activity in the starch route of synthesis.This can be the result of recessive mutation of isozygotying, or because of the technology that is commonly referred to antisense by employing or suppresses altogether or have justice downward (Sense-down) to regulate, utilizes cloned genes wild type gene to be carried out the result of part downward modulation.In addition, this female not exclusively reduces at least a specific homotype enzyme in the starch route of synthesis.This can be because the recessive mutation gene or the part downward modulation effect of heterozygosis.No matter how female produces, and it can only serve as female part.In order guaranteeing, to need to adopt one and eliminate the step that first parent produces the pollen ability in this.This method comprises with the pollen that serves as the male parent plant imparts to the plant that serves as female parent, and wherein the male parent is not mutated parent.The grain that collection is produced by described first parent.In addition, this method can comprise extract starch from grain.

The present invention also comprises having the plant that makes the gene of at least two kinds of incomplete reductions of specific homotype enzyme in the plant amylum route of synthesis.The present invention also comprises the starch that is produced by described plant, with described similar, compares but contain the starch that the genomic material plant of the isoreagent that does not form enzyme in the plant amylum route of synthesis forms, and it has changed structure.

Plant is starch as described in cereal (in cereal) forms.Produce cereal by female that for example has wax shape genotype (WxWx) and the androecy hybridization that for example has amylose enriching substance genotype (aeae), wherein the genotype of cereal endosperm is wxwxWX/AeAeae.

In other words, the present invention relates to produce starch plant, it contains and comprises the genomic material that makes at least two kinds of incomplete genes that reduce of specific homotype enzymic activity in the described plant amylum route of synthesis, by this, if described plant can produce much more starch for containing the plant that makes the gene that two kinds of identical in the starch route of synthesis specific homotype enzymic activitys reduce fully.

The endosperm of cereal of the present invention has two kinds of genes, contains the recessive mutation gene of a gene dosage and the wild type gene of two gene dosages; Wild type gene with a recessive mutation gene that contains two gene dosages and a gene dosage.Included cereal in description of the invention, the genotype of its endosperm be wxwxWX/AeAeae or

AeaeAe/WxWxwx, or wxwxWx/DuDudu, or duduDu/WxWxwx, or aeaeAe/DuDudu.

Or duduDu/AeAeae, or wxwxWx/SuSusu, or susuSu/WxWxwx, or aeaeAe/SuSusu, or

SusuSu/AeAeae, or duduDu/SuSusu, or susuSu/DuDudu

Starch of the present invention derives from the cereal that genotype is wxwxWX/AeAeae.Starch of the present invention also derives from the cereal that genotype is Aeaeae/WxWxwx.

Female of the present invention has the diploid gene type of aa/BB and the triploid genotype of aaA/BBb, and wherein a is the recessive mutation gene, and A is a wild type gene; B is the recessive mutation gene, and B is a wild type gene, so described starch is to be changed by normal starch, wherein a and b can be selected from ae, wx, sh, bt, h, su, fl, op, and A and B can be selected from Ae, Wx, Sh, Bt, H, Su, Fl, Op.

The starch that obtains according to the present invention can produce the gel of strong elastic, but the utmost point is removed in mouth rapidly.Starch of the present invention is compared with conventional starch, can form the gel with unique quality.This unique quality makes starch of the present invention be suitable as the substitute of conventional gel natural gum (as natural gum and gelatin) in whole or part food formula.Find that also starch of the present invention can produce bigger flexible gel than common starch.And find that the formed gel of corn starch that is produced by corn is improved than its transparency of gel that is formed by common starch.The transparency of this improvement is visible for human eye, and helps to provide the more foodstuff of appetizing.

Brief description of drawings

The present invention is illustrated by description and the embodiment with reference to accompanying drawing hereinafter by the mode of explanation.

Fig. 1 is the enzymic activity figure of single mutant different genes amount.

Fig. 2 a is the DSC scintigram of wax shape, amylose enriching substance and common starch.

Fig. 2 b is the DSC scintigram of double-mutant (aeaeae/wxwxwx).

Fig. 2 c is the starch DSC scintigram from middle mutant (aeaeAe/wxwxwx).

Fig. 2 d is the starch DSC scintigram from mutant (wxwxWx/AeAeae) in the middle of another.

Fig. 3 a is the Brabender datagram of common starch in various pH.

Fig. 3 b is the Brabender datagram of waxy starches in various pH.

Fig. 3 c is the Brabender datagram of 70% amylose in various pH.

Fig. 3 d is the Brabender datagram of double-mutant starch in various pH.

Fig. 3 e is the Brabender datagram of the first middle mutant starch in various pH.

Fig. 3 f is the Brabender datagram of the second middle mutant starch in various pH.

Fig. 4 a is design and the restriction enzyme site that illustrates the plant conversion carrier that is used to change branching enzyme I gene expression dose.

Fig. 4 b illustrates the design and the restriction enzyme site of the plant conversion carrier that is used to change branching enzyme II gene expression dose.

Fig. 4 c illustrates the design and the restriction enzyme site of the plant conversion carrier of amylosynthease (wax shape) gene expression dose that is used to change combination.

Fig. 4 d illustrates the design and the restriction enzyme site of the plant conversion carrier of the gene expression dose that is used to change the soluble starch synzyme.

Fig. 5 schemes at gel that starch of the present invention makes and the gel that is made by aewx starch and the modulus of elasticity (G ') in the gel comparison procedure that is made by wax shape (wx) starch.

Fig. 6 is modulus of elasticity (G ') figure that draws at the strain of starch of the present invention and aewx starch.

Detailed description of the present invention

In a broad aspect, the crop strain that the present invention relates to improve, this crop strain has been handled the expression of at least two kinds of amylosyntheases, and described enzyme can change output and the type of starch, then changes the cereal that is produced by described plant.

Have been found that the plant that contains at least two kinds of part downward modulations or reduce the gene of specific homotype enzymatic activity in the starch route of synthesis has a large amount of starch with unexpectedly producing in grain, and produce altered starch type.

It is because its endosperm that has changed that special product corn or mutant plant are different from " normally " corn. The endosperm of this variation produces the starch branch of height, or the sugared composition that changes, or different grain structures. Endosperm is become with avette by sperm, and will affect the formation of endosperm to parents' selection.

The present invention is made of two main method. Utilize the mutant breeding in selected crop varieties scope, to form the present invention. Utilize gene-transformed plant to form the present invention in each Plants, wherein said gene can partly be reduced two or more enzymes in the starch route of synthesis. Or rather, be adjusted to about 1/3 of normal enzyme activity under the activity with one of homotype enzyme in the starch route of synthesis, and another homotype enzyme is 2/3 of normal enzyme activity, perhaps two kinds of homotype enzymes in the starch route of synthesis all is adjusted downward to about 2/3 of normal enzyme activity. Every kind of method has the advantage of himself.

At first, utilize mutant in cereal crops, to develop unique cereal and starch is widely known by the people. Yet, the present invention why have characteristic and surprising be to produce the grain with normal starch property because estimate the present invention. Following table will illustrate that how unexpected the present invention is.

Table 1

The type of the starch of parent's genotype endosperm

(at first being female) genotype starch yield

Wild type AA^*AA AAA is normal

Gene dosage AA^*Aa AAa is normal

      aa ^*AA aaA is normal

Single mutation aa^*Aa aaa change reduction-

Two sudden change aa/bb^*Aa/bb aaa/bbb changes reduction

Mutant aa/BB in the middle of the expected results of the present invention^*AA/bb aaA/BBb is normal

      AA/BB ^*Aa/bb AAa/BBb is normal

      aa/bb ^*AA/BB aaA/bbB is normal

      aa/bb ^*Aa/BB aaa/bbB is normal

      aa/BB ^*Aa/bb aaa/BBb is normal

Actual result of the present invention

Middle mutant aa/BB^*AA/bb    aaA/BBb    Altered Medium to

           AA/BB ^*aa/bb    AAa/BBb    Altered high-70％

aa/bb ^*AA/BB????aaA/bbB????Altered?of?normal

aa/bb ^*aa/BB????aaa/bbB????Altered

Aa/BB ^*Aa/bb aaa/BBb Alteredaa=mutator gene (isozygotying) AA=wild type (or the homozygous gene that do not suddenly change) ^*Two interracial pollination hybridization aa/bb=of=expression two mutator genes (be and isozygoty)

Obviously, because the gene that the genotype of endosperm of the present invention does not have complete recessiveness shown in the table estimates that the output and the structure of starch will be normal.In fact, do not show that according to the present invention cereal has normal starch structure.From in history, when altered starch produces, the just minute quantity that produces usually.The starch that the present invention changes is higher than desired amount surprisingly.In addition, the production of this starch is more simply than the production of double-mutant crop.In the past, had only the single mutant hybrid to be widely used in the large-scale production of starch.And in order to develop double-mutant, two parents must have sudden change, and the latter need carry out the trial of significant research and development, and starch yield is not high as a result, and seed gelation power is bad.Therefore, it is possible having only the small-scale production double-mutant.

The present invention includes to produce and have the method for the cereal that has changed starch quality, comprise the parent that plantation can be bloomed, this parent has reduced the activity of at least a specific homotype enzyme (A) in the starch route of synthesis basically fully, and does not reduce the activity of another kind of at least specific homotype enzyme (B) in the starch route of synthesis.Another parent does not reduce the activity of a kind of homotype enzyme (A) in the starch route of synthesis, and has reduced the activity of at least a other specific homotype enzyme (B) basically fully.Then, must eliminate the ability that described first parent produces pollen, make pollination be undertaken, collect the grain that produces by described first parent at last by the described second mutant parent.In addition, method of the present invention can comprise extracts starch from grain, and described starch is used for showing the various uses of its value as special product starch.

In order to prepare colloidal sol of the present invention, can prepare the slurry of the starch of the present invention that contains water and effective dose, and colloidal sol is hotted plate to form paste.Generally speaking, the temperature of hotting plate feasible slurry rises to more than the gelatinization temperature of about starch, and makes starch carry out enough shearings, so that particle splits paste formation.All particles are all split.Preferably, the amount of the starch of the present invention that contains of colloidal sol is about 1-20% of colloidal sol gross weight.Slurry is hotted plate more than 90 ℃ about, to add the foodstuff prerequisite for thickening characteristic.The time of hotting plate is about 1O minute.If starch is processed, make it inflatable in cold water, colloidal sol then of the present invention need not hotted plate.Hot plate step and generally comprise the gelatinization temperature that the water slurry temperature of starch of the present invention is risen to described starch, and starch is sheared, particle is split, paste forms.

The colloidal sol or the thickener blend composition of starch of the present invention are added in the foodstuff in a usual manner, the advantage of starch of the present invention is provided for foodstuff.

In order to prepare the foodstuff of thickening, colloidal sol prepared in accordance with the present invention is combined with foodstuff, again said composition is hotted plate to required degree so that the thickening foodstuff to be provided.Carrying out conventional mixing makes colloidal sol combine with foodstuff.The composition of hotting plate colloidal sol and foodstuff also can carry out in a usual manner.

Can selectively be, starch of the present invention is mixed with foodstuff that the slurry that maybe will contain starch of the present invention and water mixes with foodstuff, the gained mixture is hotted plate to required degree again, obtains the foodstuff of thickening.When starch itself or starch-containing slurry itself are mixed with foodstuff, must hot plate the mixture of gained, so that the foodstuff of thickening to be provided.Finish in a usual manner and mix and hot plate.Hot plate about and carry out more than 90 ℃.The time of hotting plate is about 10 minutes, but can the amount and the different of shearing displacement of existing foodstuff change when mixing in the process of hotting plate.

This class thickener blend composition can provide great economic advantages for the user.The quality (clean breakingtexture) that those people that are familiar with prior art utilize various gel natural gum to rupture fully for a long time.Application of the present invention includes but not limited to gummy candy, gel dessert.Sugar skin and tablespread, and can substitute traditional gel natural gum, as κ angle fork algae, agar, pectin or gelatin.Yet these conventional gel natural gum can be very expensive, and exist comprise sick, lack heat or absolute acid stability, limited availability or lack other disadvantages such as approval of Jewish canon.Have been found that starch of the present invention can substitute all or part of said conventional gel natural gum.

For the gel natural gum in the substitute food product prescription, the weight ratio of operable starch of the present invention and gel natural gum is about 1: 1.The starch in replace gel natural gum of the present invention of available greater or lesser amount.This class gel natural gum comprises gelatin, pectin, angle fork algae, gum Arabic, bassora gum, guar gum, locust bean gum, Zanthan, agar, ammonium alginate and CMC.

Under the nature situation, starch of the present invention can be used for any food formula, as long as need provide the gel feature and thoroughly for example cracked from mouth, starch of the present invention can be used for using before this in the food formula of common starch, food with improved characteristics is provided by this, and promptly comparing with the identical food formula that utilizes common starch can thoroughly cracked (clean break).

Thorough fracture with the gel of starch of the present invention preparation is applicable to various food.All valuable in the application that thoroughly breaks at various bakery products of starch gel, for example be used for the cream or the fruit filler (as lemon, banana fat or Bavaria fat) of pie and the high solid water core of low fat that is used for cooky.Starch of the present invention can also improve for example quality of fig bar shaped cake (fig bar) and meat jelly of mousse, custard, jam dumpling.

What used term starch was represented in this specification and claim is not only the pure substantially starch granules that extracts from starch yielding plant, and comprises the cereal product of starch granules, as flour, the cereal of shelling, cornflakes and meal.

Embodiments of the invention given below are just in order to reach illustrational purpose.These embodiment are not intended to limit type of the present invention or purposes.The present invention or its cereal or its starch or its sugar can be used for but is not limited to the preparation of foodstuff, paper, plastics, adhesive, coating and the production of alcohol and corn syrup product.Embodiment 1

The physical property of the various embodiments of the present invention (similar genotype is from different corn hybridizations).Shown in each table shown the data of evaluation novel starch well known to those skilled in the art.About moisture, oil, protein, DDGS and starch percentage data are used to estimate the productive rate and the potentiality of milling.Starch DSC (differential scanning calorimetry) data are useful aspect the culinary art of evaluation starch and gel characteristic.The data of starch granules size are used to determine milling and stalling characteristic of starch.Brabender and gelatinized corn starch data are epochmaking for estimating novel starch in the ability that improvement is starved of in the food applications of specific starch thickening, gelatinization and gel characteristic.Whether these class data can make that when being interpreted as collected complete information those skilled in the art determines, need the characteristic and the potentiality of starch are carried out more deep detailed test.

Genotype	% moisture	% starch	% protein	% oil	The % DDGS
						?aeae/wx	????8.01	??72.12	??11.30	??3.63	??6.51
?aeae/wx	????8.00	??71.34	??11.74	??3.79	??6.41
						?aeae/wx	????8.03	??67.52	??12.61	??3.67	??7.01
?aeae/ae	????8.13	??60.11	??14.59	??6.60	??10.14
						?su1su1/wx	????8.18	??69.66	??12.09	??4.03	??6.91
?dudu/wx	????7.98	??71.10	??11.57	??3.92	??6.61
						?dudu/su1	????7.90	??70.53	??11.67	??3.93	??6.81
?flfl/o	????7.78	??70.23	??11.60	??4.23	??6.59
						?su1su1/du	????7.81	??69.00	??12.20	??4.77	??7.98
?su1su1/ae	????7.88	??69.03	??12.27	??4.88	??8.02
						?su1su1/su2	????7.95	??69.67	??12.28	??4.55	??7.49

Table 2-Com Data

S93 % % % % %Entry background Row moisture starch protein oil DDGS

1????aeaewx?????4660????9.15????64.80????14.58????4.47????7.64

2????aeaesu?????4636????9.27????66.25????14.77????4.54????7.40

3????aeaedu?????4612????9.24????66.01????14.65????4.73????7.80

4????wxwxae?????4600????9.07????65.72????13.91????4.38????8.14

5????wxwxsu?????4648????9.06????66.03????14.79????4.08????7.83

6????wxwxdu?????4624????8.77????65.32????15.62????4.27????8.23

7????susuae?????4594????8.85????64.83????13.11????4.71????8.49

8????susudu?????4672????8.73????65.39????13.20????5.01????8.35

9????susudu?????4618????8.77????65.15????14.52????4.35????8.26

10???single?ae??4582????9.14????60.52????15.19????5.95????9.29

11???single?wx??4654????8.83????65.44????14.04????4.77????9.43

12???single?su??4630????7.75????54.98????14.25????6.50????9.78

13???duduae?????4588????8.97????69.26????12.30????4.89????6.85

14???duduwx?????4666????8.88????70.43????11.25????4.40????6.96

15???dudusu?????4642????9.48????70.43????13.48????4.67????6.92

16??single?du???4606????8.50????65.54????12.02????6.03????9.33??-Starch?Data

S93 % % % % % Entry background Row moisture starch protein oil amylose L-max

1????aeaewx????4660????3.82????86.44??1.68????0.18????31.60????597.1

2????aeaesu????4636????6.51????86.95??1.56????0.20????31.74????603.9

3????aeaedu????4612????5.22????86.44??1.70????0.11????33.38????598.8

4????wxwxae????4600????7.33????87.89??0.39????0.09????21.10????601.1

5????wxwxsu????4648????7.27????85.87??1.32????0.09????24.68????591.7

6????wxwxdu????4624????7.31????86.07??1.12????0.10????24.57????595.2

7????susuae????4594????8.19????86.32??0.93????0.07????29.00????604.5

8????susudu????4672????8.09????87.16??0.52????0.08????27.86????599.6

9????susudu????4618????7.67????84.20??0.60????0.12????30.82????601.1

10???single?ae?4582????7.16????83.31??1.37????0.15????65.55????602.1

11???single?wx?4654????8.30????85.16??0.51????0.14????0.43?????xx

12???single?su?4630????6.82????80.92??5.08????xx??????29.32????598.4

13???duduse????4588????6.58????84.31??0.62????0.17????29.86????xx

14???duduwx????4666????11.08???84.77??0.48????0.16????28.74????xx

15???dudusu????4642????8.48????87.34??0.62????0.16????26.07????xx

16???single?du?4606????8.36????80.70??0.46????0.08????37.88????608.9

Table 2-(starch)

％ ％ ％ ％ ％Entry L-max 1 ae ae/wx 9.56 83.84 0.63 0.05 29.44 593.1 2 ae ae/wx 7.96 85.94 0.70 0.05 27.89 596.6 3 ae ae/wx 11.71 82.54 0.82 0.05 42.54 595.1 4 ae ae/ae 6.09 84.17 1.24 0.14 63.60 602.4 5 su1 su1/wx 4.37 90.00 0.56 0.09 28.36 600.0 6 du du/wx 5.95 88.50 0.61 0.09 27.98 596.9 7 du du/su1 6.93 82.66 0.69 0.10 29.98 600.8 8 fl fl/O 9.24 82.72 0.93 0.14 29.29 596.8 9 su1 su1/du 9.01 84.82 0.71 0.02 29.57 600.8 10 su1 su1/ae 7.78 84.81 0.63 0.03 29.90 600.8 11 su1 su1/su2 7.61 85.02 0.49 0.02 29.68 602.3 12 White waxy 18.34 94.94db 0.77 0.09 2.74 526.9-Starch DSC Data

Entry ℃ J/g ℃ ℃ ℃ 1 ae ae/wx of genotype Peak Delta H Peek II Onset Endset 67.7 11.67 100.0 62.6 76.6 2 ae ae/wx 66.5 10.33 96.8 61.9 74.8 3 ae ae/wx 74.6 10.00 100.8 68.8 83.4 4 ae ae/ae 81.0 11.83 xx 66.0 107.4 5 su1 su1/wx 69.7 12.00 98.3 64.2 77.7 6 du du/wx 72.2 11.50 99.5 66.6 82.3 7 du du/su1 70.2 9.67 98.4 65.7 78.3 8 fl fl/O 71.4 12.33 100.9 67.1 80.7 9 su1 su1/du 68.8 7.83 99.5 63.2 79.1 10 su1 su1/ac 68.1 10.33 99.8 61.9 78.3 11 su1 su1/su2 67.4 12.00 96.1 61.3 79.5 12 White waxy 72.7 15.33 xx 66.1 82.6-Starch DSC Data

Row ℃ J/g ℃ ℃ ℃ of background S93 Peak Delta H Peek II Onset Endset Entry

1????aeaewx????4660????73.8????11.17????99.4????67.7????82.8

2????aeaewx????4636????73.0????11.50???101.0????66.6????83.4

3????aeaewx????4612????73.3????10.83????98.6????66.7????83.0

4????wxwsae????4600????72.9????12.83????99.9????67.4????81.7

5????wxwssu????4648????73.7????12.17????98.2????68.2????81.6

6????wxwsdu????4624????73.6????11.33????99.6????69.3????80.5

7????susuae????4594????72.0????10.33????97.7????67.2????79.7

8????susudu????4672????71.9?????9.83????98.9????67.0????79.1

9????susudu????4618????72.5????10.33????97.0????68.4????79.8

10???single?ae?4582????84.6????15.67????xx??????68.7???106.9

11???single?wx?4654????72.5????16.17????xx??????68.1????81.4

12???single?su?4630????70.9????15.50???100.8????63.6????77.2

13???duduae????4588????71.8????13.50???100.5????67.1????81.1

14???duduwx????4666????71.9????10.17????98.8????67.2????79.5

15???dudusu????4642????71.7????10.17????97.2????66.8????79.0

16???single?du?4606????71.6?????9.50???100.1????65.8????79.8

Table 2-Brabender Data

S93?????????????????IR??????HP?????HF?????CP?????CF?Entry?Background??Row????Brabender????℃??????BU?????BU?????BU?????BU??1????aeaewx??????4660??460g/5.5％????65.0????315????285????940????940??2????aeaewx??????4636??460g/5.5％????81.5????225????225????470????430??3????aeaewx??????4612??460g/5.5％????84.5????180????180????410????380??4????wxwsae??????4600??460g/5.5％????74.0????360????355????590????460??5????wxwssu??????4648??460g/5.5％????74.0????325????325????540????470??6????wxwsdu??????4624??460g/5.5％????80.0????315????270????480????425??7????susuae??????4594??460g/5.5％????77.0????270????270????610????585??8????susudu??????4672??460g/5.5％????77.0????295????295????785????610??9????susudu??????4618??460g/5.5％????77.0????295????295????620????620??10???single?ae???4582??460g/12％?????90.5????170????170????240????240??11???single?wx???4654??460g/5.5％????68.0????750????360????415????405??12???single?su???4630??90g/5.5％?????93.5????35?????35?????40?????40??13???duduae??????4588??460g/5.5％????83.0????225????225????545????520??14???duduwx??????4666??460g/5.5％????86.0????245????245????600????550??15???dudusu??????4642??460g/5.5％????84.5????270????270????620????585??16???single?du???4606??460g/5.5％????89.0????70?????70?????160????160

IR＝initial?rise

CP＝cooling?peak

CF＝cooling?final-Brabender?Data

IR????????HP?????HF?????CP????CF?Entry????Genotype??????Brabender????℃????????BU?????BU?????BU????BU??1???????ae?ae/wx??????460g/5.5％???83.0??????220????220????520???460??2???????ae?ae/wx??????460g/5.5％???89.0??????220????220????540???510??3???????ae?ae/wx??????460g/5.5％???84.5??????270????270????510???450??4???????ae?ae/ae??????460g/12％????90.5??????490????490????1220??845??5???????su1?su1/wx????460g/5.5％???80.0??????250????240????595???535??6???????du?du/wx??????460g/5.5％???83.0??????280????250????560???470??7???????du?du/su1?????460g/5.5％???50.0/83.0?230????230????575???535??8???????fl?fl/O???????460g/5.5％???81.5??????280????255????630???560??9???????su1?su1/du????460g/5.5％???84.5??????200????200????500???460??10??????su1?su1/ae????460g/5.5％???86.0??????205????205????455???415??11??????su1?su1/su2???460g/5.5％???84.5??????180????180????420???385??12??????White?waxy????90g/5.5％????68.0??????830????220????310???270

Table 2-Starch Particle Size Data

(Volurne?Distribution)

S93??????????????????????????????????％StarchEntry?Background???Row?????Mode?um??Mean?um??Median?um??Recovery??1????aeaewx??????4660????16.90????12.57????16.04??????45.9??2????aeaewx??????4636????16.63????11.82????15.23??????57.9??3????aeaewx??????4612????16.36????11.48????15.20??????52.4??4????wxwsae??????4600????17.43????11.62????16.53??????41.5??5????wxwssu??????4648????17.72????12.43????16.59??????58.8??6????wxwsdu??????4624????17.48????12.22????16.26??????54.1??7????susuae??????4594????16.36????11.96????15.66??????55.5??8????susudu??????4672????16.65?????9.83????15.29??????58.7??9????susudu??????4618????16.36????11.82????15.61??????55.9??10???single?ae???4582????13.09?????9.07????12.35??????64.0??11???single?wx???4654????18.03????11.99????16.85??????55.7??12???single?su???4630?????7.43?????5.16?????7.95???????5.6??13???duduae??????4588????16.90????11.01????15.37??????50.9??14???duduwx??????4666????17.19????11.85????16.21??????52.5??15???dudusu??????4642????17.17????12.22????16.01??????44.9??16???single?du???4606????14.41????9.77?????13.19??????58.9-Starch?Particle?Size?Data

(Volurne?Distribution)

％StarchEntry??Genotype??????Mode?um??Mean?um??Median?um??Recovery??1????ae?ae/wx???????16.63????10.90????15.70??????51.4??2????ae?ae/wx???????16.12????10.44????14.92??????52.8??3????ae?ae/wx???????15.60????10.55????14.60??????59.4??4????ae?ae/ae???????11.53?????8.40????11.30??????63.7??5????su1?su1/wx?????14.88????10.24????14.21??????52.5??6????du?du/wx???????16.38????10.73????15.12??????52.0??7????du?du/su1??????15.87????11.03????14.98??????53.2??8????fl?fl/O????????16.12????10.73????15.35??????69.3??9????su1?su1/du?????15.87????10.73????14.92??????53.7??10???su1?su1/ae?????15.11?????7.85????14.35??????48.5??11???su1?su1/su2????15.85????10.33????14.20??????58.4??12???White?waxy?????17.19?????8.45????14.81??????71.5

Table 2-Starch Paste Data

Brookfield?????????????????Freeze

Viscosity????Gel-24hr?????ThawEntry??Genorype??????CPS.20?rpm?????(grams)?????Cvcles??1????ae?ae/wx????????6,200?????????166.6???????0??2????ae?ae/wx????????6,700?????????227.2???????0??3????ae?ae/wx????????8,000?????????256.3???????0??4????ae?ae/ae????????14,250????????111.1???????0??5????su1?su1/wx??????6,800?????????173.4???????0??6????du?du/wx????????6,000?????????141.4???????0??7????du?du/su1???????7,100?????????215.4???????0??8????fl?fl/O?????????7,500?????????209.4???????0??9????su1?su1/du??????6,300?????????219.7???????0??10???su1?su1/ae??????6,000?????????172.2???????0??11???su1?su1/su2?????5,500?????????158.8???????0??12???White?waxy??????1,900?????????15.3????????3-Starch?Paste?Data

Brookfield????????????Freeze

S93?????Viscosity?????????????ThawEntry??Genotype?????Row?????CPS.20rpm???Gel-24hr??Cvcles??1????ae?ae/wx?????4660????25,000??????151.8?????0??2????ae?ae/wx?????4636????6,500???????240.1?????0??3????ae?ae/wx?????4612????3,150???????216.6?????0??4????ae?ae/ae?????4600????5,600???????159.5?????0??5????su1?su1/wx???4648????6,100???????126.4?????0??6????du?du/wx?????4624????5,700???????114.4?????0??7????du?du/su1????4594????8,700???????254.8?????0??8????fl?fl/O??????4672????12,000??????272.8?????0??9????su1?su1/du???4618????10,250??????233.6?????0??10???su1?su1/ae???4582????2,500???????53.7 ^*????0??11???su1?su1/su2??4654????3,100???????13.3??????0??12???White?waxy???4630????275?????????29.0??????3??13???duduae???????4588????7,200???????226.5?????0??14???duduwx???????4666????7,300???????233.4?????0??15???dudusu???????4642????5,100???????332.9?????0??16???single?du????4606????1,650???????99.3??????0

^*Came out as a plug definition: differential scanning calorimetry (DSC)

IR represents initial rising

HR represents to add thermal spike

HF represents to heat terminal point

CP represents to cool off the peak

CF represents to cool off terminal point Brookfield viscosimeter

The Brookfield viscosimeter detects the shear strength (cP represents with centipoise) and the stability of gelatinized corn starch.Brabender Visco-Amylo-Graph data

Gelatinization point is represented the temperature that gelatinized corn starch forms.

Peak viscosity is represented the required temperature of gelatinized corn starch that provides available.

The viscosity of 95C represents to be easy to starch cooking.

The viscosity of 50C represents that hot gelatinized corn starch cooling procedure medium viscosity falls back.

The viscosity of 50C is represented the stability of starch cooking paste after 1 hour.

Protein in the corn, starch, oil and moisture percentage

The percentage of oil, starch and protein has reflected the mensuration to the recyclable output of starch in the corn.Protein in the starch, starch, oil and moisture percentage

The percentage of oil, starch and protein has reflected the mensuration to the purity of starch degree in the starch, and has shown milling property (millability).The percentage of amylose and L-MAX

These data provide the testing result to apparent amylose level in the starch.The starch granules size data

The starch granules size has shown starch productive rate and the recovery ability after milling processing.The shorthand record literal

Aeaewx in the table 2 represents aeaeAE/wxWxWx, and similarly duduwx represents duduDU/wxWxWx.All do not list wild type in the whole table.

Fig. 1 is the enzymic activity figure of each gene dosage (for example MMM, mMM, mmM, mmm) of allelomorph of the single mutant sudden change of amylose enriching substance and dim proterties.These data show the activity of following enzyme: sucrose synthase (ss), UDP-glucose pyrophosphorylase (UDPG-PP), glucokinase (GK), fructokinase (FK), phosphoglucomutase (PGM), phosphogvlucoisomerase (PGI), ATP dependence phosphofructokinase (PFK), PPi dependence phosphofructokinase (PFP), ADP glucose pyrophosphorylase (ADPG-PP), soluble starch synzyme (SSS), branching enzyme (BE) and associativity amylosynthease (BSS).Enzymic activity is to represent with respect to the percentage of wild type contrast (MMM).Under the situation of full mutation body (mmm), there is remarkable influence in the expression of various enzymes in the starch route of synthesis.And under the situation of fractional mutant (mMM and mmM), expression is not almost produced and can detectedly change.These data show, are that several enzymes are crossed the result of enzyme who expresses and eliminate the allelomorph coding of sudden change with the change of the viewed starch quality of single mutant.Combine by other amount (for example AeAeae) with two sudden change amounts (for example wxwxWx) and another sudden change, the ground, situation lower part that will can't see overexpression in all the other approach reduces the activity of two kinds of enzymes.

Fig. 2 is the DSC scintigram of starch, and these starch are to extract from the grain that derives from wax shape, amylose enriching substance and common (wild type) corn.This class DSC scanning result can provide mass data (referring to the data of the relevant peak temperature shown in this paper table, Δ H, peak II temperature, initial temperature and final temperature) for those skilled in the art.It should be noted that especially the high starch figure of amylose content is different from common starch and waxy starches.

Fig. 2 b is the DSC scintigram of the starch that extracts from the grain that derives from double-mutant (aeaeae/wxwxwx) corn.This class DSC scanning result can provide mass data (referring to the data of the relevant peak temperature shown in this paper table, Δ H, peak II temperature, initial temperature and final temperature) for those skilled in the art.Figure that it should be noted that double-mutant especially be different among Fig. 2 a about common starch and single mutant, wax shape and high amylose starches starch provided.

Fig. 2 c is the DSC scintigram of the starch that extracts the grain of mutant (aeaeAe/WxWxwx) corn in the middle of derive from, and this class scanning result can provide mass data (referring to the data of the relevant peak temperature shown in this paper table, Δ H, peak II temperature, initial temperature and final temperature) for those skilled in the art.The scintigram of mutant starch is different from double-mutant starch in the middle of it should be noted that especially, but similar like with waxy starches.

Fig. 2 d is the DSC scintigram from the starch of the grain extraction that derives from middle mutant (wxwxWx/AeAeae) corn.This class scanning result can provide mass data (referring to the data of the relevant peak temperature shown in this paper table, Δ H, peak II temperature, initial temperature and final temperature) for those skilled in the art.Scintigram that it should be noted that this centre mutant starch especially is different from double-mutant starch, but similar like with waxy starches.

Fig. 3 a is the Brabender datagram that derives from common starch under neutrality or acid condition.It is destroyed substantially that conventional corn starch is presented under the acid condition that is utilized viscosity.

Fig. 3 b is the Brabender datagram that derives from wax shape corn under neutrality or acid condition.Under neutrallty condition, the sudden change of wax shape influences the viscosity of starch the most especially.

Fig. 3 c is the Brabender datagram that derives from amylose enriching substance (70% amylose) starch under neutrality or acid condition.Under acidity or neutrallty condition, containing the many starch of amylose has increased viscosity.

Fig. 3 d is the Brabender datagram that derives from double-mutant (aeaeae/wxwxwx) starch under neutrallty condition.Although double-mutant starch is the wax shape sudden change of isozygotying, but still keeps original viscosity.

Fig. 3 e is the Brabender datagram of mutant (aeaeAe/WxWxWx) starch in the middle of deriving under the neutrallty condition.Do not increase though should be it should be noted that the amount of the new contained apparent amylose of middle mutant starch especially by these data, providing to be similar to the being seen viscosity intensity of high amylose starches mutant increases.

Fig. 3 f is the Brabender datagram of mutant (wxwxWx/AeAeae) starch in the middle of deriving under the neutrallty condition.Do not increase though it should be noted that the amount of the contained apparent amylose of middle mutant starch that this is new thus especially, providing to be similar to the being seen viscosity intensity of high amylose starches mutant increases.Embodiment 2

The present embodiment explanation has the production of the corn cereal of starch of the present invention.Utilize the traditional breeding and/or the technology of backcrossing, perhaps utilize, the corn plant of various genetic backgrounds can be transformed into the mutant gene type as method of mutagenesis through chemical substance treatment pollen.The another kind of selection is also can buy wax shape inbred strain and hybrid from a large amount of suppliers and fund seeds company.Can use any corn strain with good agronomy characteristic and high yield.In the present invention, the cereal type by mutagenesis and meticulous selection sudden change from the offspring who separates can change normal inbred strain into mutant strain.Described method be well-known to those skilled in the art (referring to for example Neuffer, M.G.and Chang, the induced mutation Vortr.Pfalzenzuchtg.16 in the research of M.T.1989. biology and agronomy, 165-178).Any have the closed crossing strain of commercial value all to can be used for this method.Confirm that by allelic test described strain is loaded with interested sudden change, this strain can be hybridized with known mutant strain in described allelic test, and this method is well known to those skilled in the art.And outward appearance and iodine that the grain of this strain should have typical selected sudden change dye characteristic, and this method is well known by persons skilled in the art.In order from plant, to obtain maximum output, preferably next carry out two interracial hybrid hybridization of closed crossing, described two closed crossing kinds have identical sudden change (for example be wax shape or amylose and augment formulation).Two hybrids of preferred generation, one is male, and isozygotys for a kind of sudden change, another is female, and isozygotys for other sudden change.Female and female hybrid can constitute identical or different genetic background, and having similar field maturity (promptly needing similar heat unit from gelation to long must the distribution with pollen) is crucial for described two strains.In order to make the middle mutant in field hybridize, need eliminate the generation of pollen from female.This can carry out through several different methods, includes but not limited to that the manual pollination acts on, artificial and machinery castration, and inheritance or kytoplasm male sterile are infiltrated female, imported male sterile and use chemical gametocide by genetic transformation.The genotype that this hybridization cereal contains the present invention is the endosperm of aaA/BBb, derives from this genotypic starch and is referred to as middle mutant starch.For optimum starch quality, it is well known to those skilled in the art improving its genetic background this point as much as possible.Embodiment 3

Can use multiple diverse ways that starch is extracted from cereal.Method in common is included as public " humidity " method of world wide.Its basic principle comprises soaking and separates with starch.The committed step of this method is softening cereal in the dipping flow container, and this process is optimized design, so that reach the suitableeest separation of corn grain composition.Utilize this method from the middle mutant cereal of embodiment 2 exploitations, to extract starch (wxwxWx/AeAeae).Plumule is easy to intactly spin off, and does not contain endosperm and shell.Macerate endosperm in water, its starch is easy to separate with white coagulum, and glutelin obtains with yellow grumeleuse.Place the jar that holds 50-90 metric ton cereal usually to flood 30-40 hour cereal in 48-52 ℃.Steep water contains 0.2% sulphur dioxide (SO ₂Gas charges into bubble), therefore be subacidity (pH4.0).Sulphur dioxide helps decomposing protein matrix, makes endosperm matrix be cracked into particle.Behind the dipping, with the cereal shape that grinds roughly or reduce to pulp.The plumule that is rich in oils is floating to the surface, and the endosperm of intensive starch then sinks.Finish separation by hydrocyclone (partition method continuously).After milling by " impact grinder that is called the Entoleter cracker ", starch is further purified, and described cracker impacts ring subsequently in addition and impacts by the broken slurry of hardened steel alloy sheets merchant speed of reversion type fluting.By two parts of centrifugal formation, i.e. protein (70% protein) and starch (2% protein) and the starch that takes off fibrous root is separated with glutelin, processing temperature maintains more than 45 ℃, to prevent microbial reproduction.Be heated to the flash drying method dry starch of 200-260 air-flow by injection.

Mutant cereal have the starch of purifying and preparation in this way in the middle of various, are applicable to various food, feed and industrial use.It can unmodified corn starch directly use.Also can modify by the chemistry or the physical treatment that keep grain structure, can washing granule to remove remaining reactant, use blanching step to produce ultrawhite starch sometimes.This starch can be through high temperature treatment and gelation is directly sold as gelling starches.This kind of starch can and be dry through chemical modification.Polymer itself can be produced maltodextrin or glucose by partially or completely hydrolysis.This class product can pass through fermentation and further modification, and the alcohol with generation gasoline industry perhaps is transformed into glucose the sorghum candy corn syrup that is used for sweetener industry.Embodiment 4

The sudden change that is called shrinkage-2 (sh2), crisp-2 (bt2), dim (du), sugar (su), wax shape (wx) and amylose enriching substance (ae) the homotype enzyme of following enzyme of encoding: ADP glucose pyrophosphorylase, debranching enzyme, soluble starch synzyme, associativity amylosynthease and branching enzyme.

Shrinkage-2 ADP of coding-unit glucose pyrophosphorylase.

Crisp-2 ADP of coding-unit glucose pyrophosphorylases.

The amylosynthease of the coding particle combination of wax shape.

Amylose enriching substance coding homotype branching enzyme.

Dim the changed homotype insoluble starch synzyme and the expression of branching enzyme.

Contain the change insoluble starch synzyme of sugar and the expression and the activity of debranching enzyme.

Utilize known mutant and gene dosage crossing scheme, we have detected and have been changed the influence (referring to accompanying drawing) that expression of gene is stored starch in the cereal.Utilize the bt2 mutant, we observe detectable ADP glucose pyrophosphorylase activity and are gradual forfeiture, and the synthetic minimizing of starch has good correlation in itself and the cereal.By these data can not quantitatively this enzyme produces the control intensity that flows to starch.In fact, we studies show that, this enzyme is one of main decisive in the starch building-up process, and almost can not control the synthetic speed of starch.This sudden change is provided with and changes starch structure significantly.When having when containing sugar, dim, the wax shape and the amylose enriching substance genotype sudden change, we have detected the change (the chain length change of branch and the variation of amylose and amylopectin ratios) of starch extract fine texture at present really.Under this class situation, for the control that flows to starch littler (except that sweet corn is genotypic contains the sugar mutant with being used to prepare).Under these all situations, the ratio of amylosynthease and branching enzyme all changes, thereby causes the variation of starch extract fine texture.The new result of resultant accident has found that not only sudden change can reduce the expression of key enzyme in these researchs, but also can induce the overexpression of other enzyme in the route of synthesis.In addition, only we have observed the variation of starch extract fine texture in complete mutant (mmm) genotype, and this shows that structural change only occurs under the forfeiture and the situation with the overexpression of homotype enzyme of homotype enzyme.When not wishing to be retrained by this proposal, these data declaration starch structures are not only reduced a kind of influence of expression (for example using antisense constructs) of enzyme, but also are subjected to the influence of the expression (for example with adopted construct is arranged) that increases simultaneously.

The plant conversion carrier available standards technique construction that is used for the inventive method.Because described enzyme is positioned the amyloplast part of cell, so gene construct need exist the amyloplast translocation peptide to guarantee its correct location in amyloplast.Transformation construct can the part sense orientation or is carried gene with antisense orientation.Expressing said gene causes being known as by prior art the reduction of the expression of enzymes that the effect of " have justice is common to be suppressed or antisense (sense cosuppression or antisense) " causes in plant.When only needing to reduce expression, do not need translocation peptide.Yet when needing the overexpression of enzyme, correct plastid hits target (targeting) sequence to be needed in construct, and key enzyme required for the present invention comprises the amylosynthease of branching enzyme and solubility and combination.Branching enzyme [1,4-α-D-glucan: 1,4-α-D-glucan 6-α-D-(1,4-α-D-glucan (glucano)) transferase] amylose can be changed into amylopectin (1,4-α-D-dextran chain fragment is transferred to the primary hydroxyl on the similar dextran chain), this enzyme is sometimes referred to as the Q enzyme.The soluble starch synzyme [ADP glucose: 1,4-α-D-bextran 45-α-D-glucosyltransferase] extend amylopectin, may also have the chain length of amylose.In conjunction with amylosynthease [ADP glucose: 1,4-α-D-bextran 45-α-D glucosyltransferase] extend amylose, may also have the chain length of amylopectin.

For any antisense or for having that justice is common and suppressing to make up, only need in genetically modified plants, represent partial cDNA Cloning.When the overexpression that needs enzyme, then need to express full length cDNA clone.Someone has studied the sequence (Baba, T., et al., Plant Physiology.103.565-573,1993) of corn branching enzyme I.Fisher, people such as D.K. (Fisher, D.K., Boyer, C.D. and Hannah, L.C.Plant Physiology.102:1045-1046,1993) have studied the Q-enzyrne II of corn embryosperm.Other has article to report the relation (Mu, C., et al., Plant Journal 6,151-159,1994) that has shown between 76KDa polypeptide and corn (cv B73) endosperm soluble starch synzyme I activity.The corn wax shape gene locus of coding UDP-glucoamylase glucosyltransferase clone in 1986 (Kloesgen, R.B.et al., Mol.Gen.Genet.203,237-244).Recently, James, M and Wright, A. utilize the method for transposon mutagenesis gene location to observe the sequence of corresponding primverose gene locus (The Plant Journal).It is believed that the gene of any this proteinoid of coding all relates to debranching enzyme, and can be used for construct of the present invention.

It is believed that chloroplast shifts peptide and has similar sequence (Heijne et al., Plant Mol BiolReporter, 9 (2): 104-126,1991).Other effective translocation peptide comprises ADPG pyrophosphotransferase (Plant Mol Biol Reporter, 9:104-126,1991), the little RUBISCO of subunit, acetolactate synthestase, glyceraldehyde-3P-dehydrase and nitrite reductase.For example, the common sequences from the many genotypic little RUBISCO of subunit translocation peptides is:

MASSMLSSAAVATRTNPAQASM?VAPFTGLKSAAFPVSRKQNLDITSIAS

The sequence that the little RUBISCO of the subunit translocation peptide of NGGRVQC corn has is:

MAPTVMMASSATATRTNPAQAS??AVAPFQGLKSTASLPVARRSSRSLGN

VASNGGRIRC. the translocation peptide sequence of the leaf amylosynthease of corn is:

MAALATSQLVATRAGLGVPDAS?TFRRGAAQGLRGARASAAADTL

SMRTASARAAPRHQQQARRGGR FPSLVVC. embodiment 5

Since nineteen ninety, people are carrying out the production of sterility rotaring gene corn plant always.Although known a large amount of DNA transmission system, but still selection is microparticle bombardment.As mentioned above, various maize mutant body gene constructs can obtain from the preservation mechanism of US and European.Shown in the accompanying drawing 4a-4d is some new examples in these constructs.Fig. 4 c has shown CaMv (cauliflower mosaic virus) promotor, wax shape gene, Adhl, no (ncpoline) but and as the pat gene and the amp of selected marker, Fig. 4 d is similar, but has shown the first homotype gene of soluble starch synzyme in the construct.Fig. 4 a also has identical construct, but has shown the branching enzyme first homotype gene.Fig. 4 b shows second branching enzyme, the second homotype gene.Certainly, also can obtain with corn breeding in other relevant construct of gene mutation body of using.

Present embodiment is with reference to figure 4c, wax shape gene construct.The purpose of this experiment is to form the closed crossing strain with part downward modulation wax shape gene action.If selected closed crossing strain has been the ae mutant, then will be by the cereal that is produced with nonmutationed closed crossing incross in the middle of mutant cereal.According to the intensity difference of downward modulation effect, female closed crossing cereal will with the mm of starch and cereal ^*/ mm ^*Type or mm ^*/ m ^*Type is similar.Very clear, transformation make to produce the more accurate downward modulation mode of action of starch synthesizing activity, so that the change of starch seems and coordinates more.

Reasonable for the degree that guarantees wax shape gene downward modulation effect, the target tissue of transformation is immature zygote plumule, can also use the callus of whole embryogenesis.The A188 plant immature zygotic embryos bud that can select is after with B73ae closed crossing strain pollination 12 days.The medium of callus be 6mM L-proline, 2% (w/v) sucrose, 2mg/ ml 2,4 dichlorophenoxyacetic acid (2,4-D) and 0.3% (w/v) Gelrite (Caroline BiologicalSupply) (pH6.0).Cultured calli, and beginning suspension culture.

Liquid nutrient medium based on MS contains 100mg/l inositol, 2mg/l 2,4-D, 2mg/l 1-naa (NAA), the 6mM proline, 200mg/l Agavain (DifcoLaboratories), 3% (w/v) sucrose and 5% (v/v) Sucus Cocois (DifcoLaboratories) are (pH6.0).Cell suspension is maintained at 28 ℃ in containing the 125ml conical flask of above-mentioned medium, in the dark shake in the revolution shaking table with 125Mom and educate.

The conversion carrier of screening Fig. 4 c, this plasmid contains 355-ladh-pat no 3 ' can select expression casette.

With cell suspension screening, be suspended from then in the 5ml suspension culture base, and place on the filter paper by vacuum tube.As be known in the art, construct is wrapped in the particulate.Bombardment is dull and stereotyped then.Again cell is changed over to the N-6 medium, and at 14 days after 1mg/l selects transformant.Cell is suspended from contains 6% (w/v) (Sea-Plaque; FMC) in the medium, and place 37 ℃.

2-5 shifts out the callus of growth after week, and is transferred to fresh selection media surface.Plant regenerates in having the MS base medium of 6% sucrose 1g/l inositol, 1mg/l NAA (34) and 0.3% (w/v) Gelrite (pH6.0).Next, embryo gelation under illumination condition in the MS medium of 0.25mg/l NAA and 3% (w/v) sucrose.Plant growing, and move in the greenhouse.Can assess the expression in the plant then.

Plant breeding, and develop into have mutant and the downward modulation action pathway the closed crossing strain, in addition, selected closed crossing offspring can have the mutant that hybridizes on the genetically modified plants after the conversion, when genetically modified plants can form required starch when female in cereal.Embodiment 6

The gelling temperature that present embodiment explanation starch of the present invention is compared with other starch.Described gelling temperature is listed in the table below in 3.

Table 3

The starch sample amylose ^*Gelling temperature ^*

℃

1 natural conventional corn 28 71

2 natural AMY V 57 80

3 natural AMY VII 73 90

4 the present invention 21 73

5 natural aewx 25 80

6 natural wxwxwx 3 72

^*Numerical value is rounded up to integer

Sample 1 be commercially produced product (American Maize-Products Co., Hammond, Indiana).The amylose percentage of above-mentioned sample 1 and gelling temperature are the averages that the random sampling by product records.99% confidence level of amylose percentage and gelling temperature is respectively 25.9-29.3 and 68.7-72.9.

AMY V and AMY VII be merchant sell amylomaize starch (American Maize-Products Co., Aammond, Indiana).Amylose percentage shown in the last table 3 and gelling temperature are the averages that records through random sampling.99% credibility interval of AMY V and AMY VII amylose percentage is respectively 53.4-62.5 and 65.5-73.8.99% credibility interval of both gelling temperatures is respectively 72.8-84.4 and 83.1-90.8.AMY V and AMY VII grow with natural corn.

Starch sample 4 is corresponding to the present invention, and sample 5 is equivalent to US5, the average of 099,911 embodiment 1.Sample 6 is equivalent to discuss the waxy starches of selling (American Maize-Products Co.).

The method of measuring amylose percentage and gelling temperature is as follows:

Calorimetric iodine method with standard is measured amylose percentage, and wherein starch is at first used the sodium hydroxide gel, then with the reaction of iodine liquid, detects the gained sample with spectrophotometer blank in the relative 2% iodine liquid of 600nm in the 1cm pond.

Method on the corresponding handbook that provides according to manufacturer, with Mettler 300 types scanning calorimeter with 30% starch solids detection DSC gelling temperature.

Apparent by last table 3, the gelling temperature of starch of the present invention can be compared with conventional corn starch.

Embodiment 7

The present embodiment explanation is compared gel strength by the colloidal sol that aewx corn starch, conventional corn starch and wxwxwx starch make respectively by the colloidal sol of corn starch preparation of the present invention.Result of the test is listed in the table below in 4.

Table 4

Sample strength (gram)

The present invention 159.5

Conventional corn starch 225.0

Aewx corn starch 55.0

Waxy corn starch 16.0

In order to go up the glue strength test shown in the table 4,, will starch again through the Fast Heating mode and in Brabender starch viscosity analyzer, be heated to 50 ℃ by with water and the mixed colloidal sol that gets of starch.Once reaching 50 ℃, then instrument is set at 1.5 ℃/minute controllability firing rate, until reaching 95 ℃.Then sample was kept 30 minutes at 95 ℃.Next, sample is cooled to 50 ℃ with 1.5 ℃, lasts 30 fens.These colloidal sols are added respectively in 4 ounces the bottle of having inserted piston.Colloidal sol was left standstill under room temperature condition 24 hours, by mensuration piston is taken out required power from colloidal sol and measure glue intensity.

Present embodiment explanation by the colloidal sol glue intensity of the present invention's preparation can with the comparing of conventional corn starch colloidal sol.Embodiment 8

Difference between present embodiment explanation aewx starch and starch of the present invention, described aewx starch is the waxy starches that a kind of its plant has triplication wax shape gene.All starch all is corn starch.

Test the rheological behavior of all starch.Every kind of starch all uses identical method through identical process of the test.Utilization has cooled detector downwards and have a Brabender Visco-Amylo-Graph gelatinized starch particle of 750g cm tube.In the Brabender cup, the starch slurry of 5.5% initial solid is heated to 60 ℃ rapidly, gelatinization is then heated to 95 ℃ with 1.5 ℃/minute speed simultaneously.Gelatinized corn starch was kept under this temperature 20 minutes, went up sample then immediately to being preheating on 70 ℃ the measurement geometry of rheometer.Carrying out the portioning rheological behavior identifies.When sample be cooled to from 70 ℃ 25 ℃ and keep 4 hours after, detect the gel solidification section that forms (falling into the linear viscoelasticity district well) with .2 hertz and .2% tension monitoring structure.What also will detect is the fluctuation of tension force, and this will detect gelatinized corn starch or glue reacts the rheology that tension level increases, and this process is also carried out at 0.2 hertz, 25 ℃.

Utilize described technology can find obvious difference between aewx starch and starch of the present invention.Fig. 5 has shown the result that adhesive curing is analyzed.Initial modulus (G ') is lower than the starch of the present invention of aewx starch, forms structure or gel quickly, and this G ' by rapid rising is proved.Therefore, the difference of starch of the present invention and aewx starch is the speed that glue forms, although both apparent iodine binding capacities are similar.Fig. 6 has only shown starch of the present invention and aewx starch has been carried out the result that tension fluctuation is analyzed.Starch wherein of the present invention has showed expansion character, and G ' rising was proved when this was raise by tension level.Formed structure is not damaged under used tension level.On the contrary, the aewx starch structure was progressively then destroyed rapidly above 4% o'clock at used tension force.Therefore, compare with aewx starch, starch of the present invention has formed at this and has tested the glue that does not break under the used tension level.On the contrary, aewx starch has shown that extremely low time dependence structure forms, and " breaks " really under the used tension force of above-mentioned technology or destroyed.Embodiment 9

The preparation of present embodiment explanation thickener blend composition of the present invention.

Starch of the present invention is mixed with a certain amount of with water, produce slurry with 10% starch weight.The crisp flavor temperature of this colloidal sol matter.When hotting plate 10 minutes for about 90 ℃, colloidal sol forms thickener blend composition with it, and it has better transparency and more crisp quality than the similar thickener blend composition by the preparation of conventional corn starch.Embodiment 10

Present embodiment compares the mouthfeel by the glue of the glue of starch preparation of the present invention and common starch preparation.

Utilize the Brabender Visco-Amylo-Graph with common starch and starch gelatinization of the present invention.Starch is heated to 50 ℃ in the Fast Heating mode then with 5.5% solid slurrying.Utilize controllability firing rate (1.5 ℃/minute) that slurry is heated to 95 ℃, then this temperature maintenance 30 minutes.Final solid is 5.9%.Again gelatinized corn starch sample impouring fruitlet is frozen in the jar, covered, make it before analyzing, to wear out 24 hours with glassine paper.Please sample the panelist then grades to sample at following Column Properties.

At first, pass judgment on the relative hardness of both feels.

Common starch starch of the present invention

5.1?????????????6.6

Relative breaking degree, hardness and the removing speed of sample when secondly, evaluation is chewed.

Common starch starch of the present invention

Complete breaking degree 3.2 8.5

Hardness 4.4 4.5

Removing speed 2.5 4.6

These results show that the hardness of these samples is similar.Yet starch of the present invention can rupture when being chewed very up hill and dale.

And amylan of the present invention is tending towards more promptly removing from mouth than common starch glue.The glue that the consistent the present invention of thinking of panelist produces is similar to gelatin or pectin, and the mouthfeel of " cleaning " is arranged.Embodiment 11

The present embodiment explanation utilizes starch of the present invention to prepare glycocoll.

Used composition and method are as follows:

Table 5

Composition %by Weight

The present invention's (%, weight)

44/62 unmixing corn syrup 56.32

Sugar, fine granular 23.98

Water 7.73

Starch 11.80 of the present invention

Citric acid 0.07

Sodium citrate 0.08

100.00 method:

Mix all compositions, utilize conventional equipment (as the jet cooking pot) boiling to 340 then.Slurry with institute's boiling injects sugared mould again, makes it to solidify.Embodiment 12

The present embodiment explanation utilizes the present invention to make the bavarian cream pie.

Used composition and method are as follows:

Table 6

Composition % by Weight

The present invention's (%, weight)

3.5% bright full milk 72.794

Sugar, fine granular 17.586

Salt, powder 0.101

The present invention 5.410

Banana spices 0.300

Fresh yolk 3.809

100.000 method:

Except yolk, all pie filling components are mixed, and in 195 boiling 3-5 minutes.Then this composition along with stirring, constant speed is cooled to 120 °F.Next add yolk, fully the mixing mixture.Again mixture is added in the conventional pie base, before enjoying, make it to be cooled to room temperature.Embodiment 13

The present embodiment explanation is made lemon pie filler with starch of the present invention.Used composition and method are as follows: method:

The water of half is merged with sugar, boil it.With all residual componentss slurrying together, add to then in the syrup that boils.The temperature of this mixture is transferred to 200 °F, kept 2 minutes.Again mixture is injected standby pie base, make it cooling, solidify.Embodiment 14

The present embodiment explanation utilizes starch of the present invention to make chocolate mousse.Prescription in the table 8 is used to prepare the mousse mixture.

Table 8

％

Frodex?24-924????????????????39.20

Sugar (bakery product is used) 30.75

Whiptreme 3554 (Kerry composition) 12.88

Starch 9.80 of the present invention

Cocoa power, Dutch Red 7.17

(Gill?&?Duffus?Products)

Leceitreme 40 (Kerry composition) 0.20

Spices if necessary

100.00 method

Merge each composition, form uniform mixture.Usage

200g mousse mixture is mixed with 1 glass of (250 gram) milk.It was mixed 1 minute with low speed with motorized agitator.Scrape earthen bowl.Mixed at high speed 3 minutes is until becoming light and bulk.Ladle out to in the service plate with spoon, enjoyed in the preposition refrigerator 1 hour.

In order to prepare the mousse mixture, all compositions are mixed.In order to prepare mousse itself, 200g mousse mixture and 250g milk are merged, and mixed on low speed.And then the high-speed stirred mixture, make it light and bulk.Mixture was put in the refrigerator 1 hour.Promptly make light and bulk mousse in this way.

Therefore, the present invention is described with the particularity to a certain degree at the preferred embodiment of the invention.But should be appreciated that the present invention is limited by the claims of advancing to lay down a definition according to existing skill, thereby can under the creative idea situation that is wherein comprised, modify or change the preferred embodiments of the invention.

Claims (35)

1, a plant species contains and comprises that at least two kinds of specific homotype enzymic activitys produce the not exclusively genomic material of the gene of reduction in the starch route of synthesis that makes described plant.

2, according to the plant of claim 1, the starch of formation has the branched structure that is different from by being similar to the starch that the described plant of claim 1 forms, and contains the genomic material of the homotype enzyme of the starch route of synthesis that does not form described plant.

3, according to the plant of claim 2, described starch forms in cereal.

4, the cereal that is produced by plant, its genotype is mm ^*/ n ^*, wherein m is first mutant, n is second mutant, ^*Equal wild type.

5, a kind of product starch plant, the genomic material that contains the gene of the incomplete reduction of at least two kinds of specific homotype enzymic activitys that comprises in the starch route of synthesis that makes described plant, described plant wanted many with the starch that produces when by this, the starch that produces in fact of described plant reduced identical two kinds of specific homotype enzymic activitys in the starch route of synthesis such as the described gene of fruit fully.

6, a kind of cereal has the endosperm genotype that is selected from following genotype composition:

wxwxWx/AeAeae，aeaeAe/WxWxwx，wxwxWx/SuSusu，susuSu/wxWxwx，

aeaeAe/SuSusu，susuSu/AeAeae，wxwxWx/DuDudu，aeaeAe/DuDudu，

susuSu/DuDu?du.

7, a kind of cereal, its endosperm genotype contains the allelomorph of second mutant of the second kind of gene that influences starch structure of the allelomorph of a kind of gene first mutant that influences starch structure of two dosage and a dosage, that described gene can be selected from is the wax shape, the amylose enriching substance, dim, horn shape, contain sugar, shrinkage, crisp, powdery and opaque gene.

8, the starch that from have the genotypic cereal of wxwxWx/AeAeae according to claim 7, extracts.

9, the starch that from have the genotypic cereal of Aeaeae/WxWxwx according to claim 7, extracts.

10, a kind of product starch plant, have aa/BB diploid gene type and aaA/BBb triploid endosperm genotype, wherein a is recessive mutator gene, A is a wild type gene, b is the recessive mutation gene, and B is a wild type gene, and its starch by normal starch change has taken place, a and b can be selected from ae, du, wx, sh, bt, h, su, fl, op, and A and B can be selected from Ae, Du, Wx, Sh, Bt, H, Su, Fl, Op.

11, a plant species, has aA/Bb/Cc diploid gene type and aaA/BBb/CCc (with its other combination) triploid endosperm genotype, wherein a is the recessive mutation gene, A is a wild type gene, b is the recessive mutation gene, and B is a wild type gene, and starch is changed by normal starch, a and b can be selected from ae, wx, sh, bt, h, su, fl, op, du, and B and A can be selected from Ae, Wx, Sh, Bt, H, Su, Fl, Op, Du.

12, production has the method for the cereal that has changed starch quality, comprises the following steps:

A) plantation parent, it can be bloomed, and has reduced the activity of at least a specific homotype enzyme in the starch route of synthesis fully;

B) plantation second parent, it has reduced the activity of another kind of at least specific homotype enzyme in the starch route of synthesis fully;

C) eliminate the ability that described first parent produces pollen;

D) impart to described first parent of blooming with described second parent's pollen;

E) collect the cereal that described first parent produces.

13,, comprise and from described cereal, extract the starch that changes according to the method for claim 12.

14, the starch that extracts in the cereal of accessory rights requirement 4, wherein a is chosen to be identical mutant with b, and B is chosen to be identical wild type with A.

15, the starch that extracts from cereal, described cereal have the wild type of the mutant and 2 dosage of 4 dosage, make its genotype every-way (side) wild type is all arranged.

16, the starch that extracts from cereal, described cereal have the wild type of the mutant and 3 dosage of 3 dosage, make its genotype that wild type all be arranged every-way.

17, single mutant male sterile plants, this mutant is selected from ae, wx, sh, bt, h, su, fl, op, du.

18, the colloidal sol that contains water and effective dose starch, described starch extract from having the genotype plant wax shape, the wax shape, amylose enriching substance (amylose extender).

19, according to the colloidal sol of claim 18, wherein existing starch percentage by weight is about 1-20%.

20, according to the colloidal sol of claim 18, wherein said plant is a corn.

21, according to the colloidal sol of claim 18, wherein said starch is cold water soluble.

22, according to the colloidal sol of claim 18, wherein said starch is graininess.

23, according to the colloidal sol of claim 21, wherein said starch extracts from corn.

24, a kind of foodstuff contains foodstuff and as the effective dose starch of neccessary composition, described starch from have wax shape, wax shape, extract the genotypic starch yielding plant of amylose enriching substance.

25, according to the foodstuff of claim 24, wherein said starch is about 0.1-10% of foodstuff weight.

26, according to the foodstuff of claim 24, wherein said starch extracts from corn.

27, the preparation method of starch-containing colloidal sol comprises the following steps:

Formation contains the slurry of water and effective dose starch, and described starch is the wax shape, the wax shape from having, extract the starch yielding plant of amylose gene type,

The described starch of boiling makes it gelation.

28, according to the method for claim 27, about 1-20% of wherein said effective dose slurry weight.

29, according to the method for claim 27, wherein said starch extracts from corn.

30, the method for foodstuff thickening comprises the following steps:

Foodstuff is mixed with the starch of effective dose, and described starch is the wax shape, the wax shape from having, extract the genotypic starch yielding plant of amylose enriching substance,

The described foodstuff of boiling makes it thickening.

31, according to the method for claim 30, wherein said starch extracts from corn.

32, according to the method for claim 30, the amount of wherein said starch is about 0.1-10% of described foodstuff weight.

33, make improving one's methods of the foodstuff contain gelatin, described improvement comprises that the colloidal sol with claim 18 substitutes the gelatin in the described foodstuff of at least a portion.

34, making contains improving one's methods of natural gum foodstuff, and described improvement comprises that the colloidal sol with claim 18 substitutes at least a portion natural gum.

35, according to the method for claim 34, wherein said foodstuff is gummy candy, gluey dessert, sugared skin or is coated with the flavor product.

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