CN101255423A - Method for creating cultivated silkworm chromatic cocoon by using pigment protein - Google Patents

Abstract

A colorful silkworm cocoon creation method by pigment protein is disclosed. A pigment protein determining color of coral reed and related marine creature is fusion expressed in bombyx mori silk fibroin by genetic modification of silkworm and especially genetic modification for diapause varieties, so that novel colored silkworm cocoon is created. Exogenous pigment protein is fusion expressed in silk fibroin, and exists stably and uniformly in silk fibroin molecular, so pigment protein will not be lost in silk reeling procedure, and color variety of pigment protein is abundant. The method used by the invention solves two problems of little variety in color of silkworm cocoon and instability in pigment binding, simplifies spinning process of silk fabrics, eliminates harm of dyeing process to silk fiber quality and human health.

Description

Utilize the method for chromoprotein cultivated silkworm chromatic cocoon

Technical field

The present invention relates to biotechnology, bioengineering field, particularly relate to a kind of method of utilizing chromoprotein cultivated silkworm chromatic cocoon.

Background technology

Biological physical efficiency presents various colors, thereby constitutes in riotous profusion colorful biological world.The color of organism depends primarily on the factor of two aspects: tissue or special optical texture and the existence of coloring matter, i.e. schemochrome and the chemical look of organ.The proteic special space structure of domestic silkworm silk has given silk pearlescent gloss, and the existence of small molecules coloring matter then makes silk cocoon present distinct colors.The gloss of natural, graceful and poised elegance, slim and graceful comfortable sense of touch, good dress material drape and curve, moderate water-absorbent and to the inherent good characteristics such as affinity of human skin make silk quilt be described as " fiber queen " just.

Since be " queen " in the fiber, why does not silk fiber occupy the very big market share in five big fibers (cotton, fiber crops, hair, silk and regenerated fiber)? this is because except having innumerable advantage, natural silk also has some shortcomings that can't remedy at present: the cultivated silkworm breed variety that uses during 1. silkworm already produces at present mostly is white cocoon kind, before being used to weave cotton cloth, the raw silk of its reel silk from cocoons system needs to dye, and the silk staining technique is still not mature enough at present, causes specking, yellowing and embrittlement easily; 2. the essence owing to silk is protein, its thermotolerance, sun-proof poor-performing, and the chemical substances such as picric acid that use in ultraviolet radiation and the dyeing course all can cause a look yellowing.Silk with good characteristic like this but can't more comprehensively be promoted the use of because of the shortcoming of above-mentioned two aspects, no matter this to silkworm industry worker or to human beings'health, all is a no small loss and sorry.So numerous scholars of China and foreign countries are target to solve these two difficult problems, carried out discussion research in various degree from different angles.As improve staining technique, to silk albumen carry out modification etc. all be some directly and practical thinkings, but the shortcoming of the silk of all failing to overcome effectively for a long time.Another kind of feasible thinking is exactly to utilize natural colored cocoon kind or create new chromatic cocoon kind, need not print and dye behind the silk cocoon filature of acquisition and directly applies to fabric.

The commercial variety of raising almost is white cocoon kind entirely at present, and the cocoon look of silkworm is rich and varied in fact, and being broadly divided into yellow red cocoon system and green cocoon is two big classes.That yellow red cocoon system comprises is yellowish, golden yellow, yellowish pink, red, wormwood artemisia look, rust etc., its cocoon look comes from carotenoid and the xenthophylls class pigment in the mulberry leaf, the former comprises β-Hu Luobusu and newborn β-Hu Luobusu, the latter comprises xenthophylls, taraxanthin, zeaxanthin diepoxide, xanthoglobulin xanthin etc., and its pigment is distributed in the silk gum.The pigment that green cocoon is then is to come from the flavonoid pigment that generates in the body, and its pigment is distributed in silk gum and the fibroin.No matter be yellow red cocoon system or green cocoon system, the formation of its cocoon look all by the Gene Handling of one or more complexity, relate to the path of a numerous and complicated and be subjected to influence of various factors.Coloring matter will arrive in the sericterium from mulberry leaf and combine with the silk material, must be through the digestive tube of silkworm, through digesting tube wall, process blood, finally combining with fibroin or silk gum through the sericterium wall.In this process, the ability that sees through of the effect of Digestive system, midgut wall and the formation that all can directly have influence on the cocoon look through position, the oxidation capacity of blood, the translational speed of coloring matter, the perviousness of sericterium wall, the absorption site of sericterium and the binding ability of coloring matter and silk-protein etc. in the digestive tube.In addition, avoiding the complicacy of tame silk cocoon look formation does not talk, even the pigment in the mulberry leaf can successfully " be forced five passes and slay six captains " all, enter sericterium and the coloured silk cocoon of final formation, for tame silk cocoon look, still the problem that exists two keys and be difficult to remedy: 1. the kind of pigment in the mulberry leaf is limited, even they can all enter the formation that sericterium participates in colored cocoon, the color cocoon kind that forms also can only comprise all colours of yellow red cocoon system and green cocoon system at most, and these colors have only accounted for a very little part in natural all colours; 2. except the partial pigment of green cocoon is present in the fibroin; pigment in the natural colorful cocoon nearly all is present among the silk gum; and silk gum only plays the effect of protection and adhesion in silk; part runs off along with the effect of coming unstuck in silk reeling process; not only the pigment in it is to the not contribution of color of silk goods; become a kind of pollution on the contrary, this also is that people utilize white cocoon and the main reason of superseded colored cocoon in breed breeding for a long time.

Up to the present, change and the thinking of creating coloured silk cocoon comprises following several: 1. by the engineered cocoon color base of molecular genetic because of; 2. freshen the food pigment; 3. utilize existing chromatic cocoon kind to breed new color cocoon kind; 4. screen mutation.These methods can both change or the cultivated silkworm colored cocoon to a certain extent, but the chromatic cocoon of its generation all can't be broken away from that color category is limited, pigment is in conjunction with defectives that natural color cocoon had such as instabilities.

The priority of domestic silkworm gene framing figure and meticulous figure announces, for the research of domestic silkworm gene and molecular level provides a more wide platform, also injected new vigor and vitality for the development of modern silkworm industry.Especially reaching its maturity of technology such as silkworm transgenosis makes that utilizing modern biotechnology to create the new bio material becomes important topic in silkworm genome times afterwards comprehensively and the modern agricultural technology system.But object that biotechnology is studied and target are gene and protein, for general non-albumen and not have the proteic coloring matter of secure bond but be at a loss what to do.Pigment in the natural pigment of occurring in nature, the especially organism, the overwhelming majority is non-proteic materials such as alkene class, what they disperseed or concentrated is fixed on a certain tissue or the organ, makes organism painted under the condition that corresponding prothetic group exists.But, small part coloring matter exception is also arranged, it is chromoprotein, this class pigment belongs to the member of green fluorescent protein (GFP) family more, they are pure protein in itself, express by individual gene, and without any need for prothetic group, only rely on the endogenic chromophoric group of molecule and present distinct colors.This class chromoprotein is present among the relevant marine organisms of karang, bright colour is the attractive properties that relevant marine organisms such as karang comprise from the sponge to fish etc., and these bright colour all are to depend on that one or more chromoproteins are at its intravital expression.

Based on above consideration, this research invention utilizes transgenic method, the proteinic gene of the marine organisms color that above-mentioned decision karang etc. is relevant changes in the silkworm, make its special and silk fibroin amalgamation and expression in sericterium, can avoid dexterously then that general coloring matter enters the complicated path of silk-protein and be difficult to the people is that a difficult problem of controlling is created coloury chromatic cocoon.In addition, this research invention is because exogenous pigment albumen is amalgamation and expression in fibroin, can be present in the fibroin molecule to stable and uniform, chromoprotein can't run off because of coming unstuck in silk reeling process, and the color category of chromoprotein is abundant, utilizes this method can break through that the cocoon look is subject to the limit of mulberry leaf pigment and the kind of expanding tame silk cocoon look widely.

Summary of the invention

The object of the present invention is to provide a kind of route method of utilizing chromoprotein cultivated silkworm chromatic cocoon.This route method is utilized the silkworm transgenic technology, the transgenic technology of diapause breed variety especially, and the chromoprotein of relevant marine organism color such as amalgamation and expression decision karang in bombyx mori silk fibroin, thus create the coloured silk cocoon that makes new advances.

The present invention utilizes chromoprotein cultivated silkworm colored cocoon first, and has obtained the novel silk cocoon of multiple color by the silkworm transgenic technology, has especially obtained a collection of never occurred in silkworm, the chromatic cocoon that utilizes prior art also can't realize.The present invention has broken through the technical bottleneck of the man silk cocoon look of artificial control dexterously, has set up a kind of technological line method of reliably and efficiently creating the new type colorful cocoon, provides a kind of new method for utilizing modern biotechnology to create new biological source material.

The present invention utilizes chromoprotein to create the method for new type colorful cocoon, realizes by following steps successively:

(1) obtaining of chromoprotein gene: the respective organization of choosing karang or other relevant marine organism is extracted genome or synthetic relevant cDNA, amplify the gene fragment of expressing chromoprotein, perhaps directly buy the carrier that contains the chromoprotein gene to relevant biotech firm;

(2) structure of silkworm transgene carrier: according to the requirement of the public transgenic technology of silkworm at present, make up the piggyBac expression vector, the red fluorescent protein (DsRed) that starts with the promotor 3xp3 of eye and neural specific starts the chromoprotein gene that obtains with the bombyx mori silk fibroin heavy chain promoter and realizes amalgamation and expression as selection markers;

(3) silkworm embryos microinjection: with white cocoon cultivated silkworm breed variety as starting materials, the oolysis just given birth to removed after the diapause at the back 3h-6h of laying eggs reorganization transposon vector plasmid and the assistant carrier plasmid of 10-15nl, total concn 400ng/ul injected the silkworm seed of termination of diapause from silkworm seed outside of belly central authorities, and inject the aperture that stays with nontoxic glue sealing chorion with microinjection instrument;

(4) screening of transgenic silkworm: the silkworm seed that injection is finished is sterilized in the formaldehyde vapors of 35-37% behind the 5min, place under 25 ℃, the condition of relative humidity more than 85%, hasten the hatching of silkworms up to hatching, the newly-hatched silkworm that raising hatches out, the silkworm moth in the present age (G0) is carried out selfing or backcrosses, obtain G1 for silkworm seed, scanning G1 obtains the transgenosis individuality for silkworm seed, promptly obtains the transgenosis individuality of respective color silk cocoon;

(5) acquisition of colored silk cocoon transgenic strain material: with the transgenic positive individual feeding that screens to put on an arrow harvesting of cocoon and further selfing select pure, promptly obtain can genetic stability colored silk cocoon transgenic strain material.

In the above-mentioned steps, h represents hour, and min represents minute.With the method for bombyx mori silk fibroin heavy chain promoter startup chromoprotein gene fusion expression, patent applicant of the present invention applies for a patent (application number 200610054442.x) on July 14th, 2006, and is now open.

Advantage of the present invention is:

The present invention has realized utilizing chromoprotein to create coloured silk cocoon first, and the imagination that material is innovated organically combines with the silkworm transgenic technology, has broken through the technical bottleneck of the man silk cocoon look of artificial control dexterously.Advantage of the present invention is:

(1) leaf of plant, fruit be owing to contain chlorophyll, anthocyanidin, and the carotene that contains of certain plants, all be green, red, and animal is food mostly with the plant, so blue seldom appearing in the common organism.Silkworm is no exception, so silkworm has only yellow red cocoon system and green cocoon system.According to the principle of color matching, have only when having three kinds of essential tones of red, green, blue simultaneously, just can arrange in pairs or groups mutually and access various colors.The present invention utilizes chromoprotein to create blue silk cocoon, for comprehensive availableization that realizes the silk cocoon color created condition.

(2) expression-form that uses among the present invention is bombyx mori silk fibroin heavy chain amalgamation and expression, main body---fibroin---amalgamation and expression of assumed appearance element and silk, chromoprotein just can be distributed in to stable and uniform among the fibroin in sericterium secretion fibroin, pass through the squeezing crystallization of silking process again, chromoprotein makes firm the combining of coloring matter and fibroin, even can't run off because of coming unstuck in filature and raw silk purified process.With the fabric that this silk is produced, need not print and dye to directly apply to and weave cotton cloth, will simplify greatly and spin clothes technology, save a large amount of production costs.

(3) because the silk compact structure, though be easier to paintedly at dyeing time ratio, dyestuff is not very stable with combining of silk, causes the decolouring of silk fabrics and the phenomenon that fades easily.And the chromoprotein among the present invention is to merge completely with fibroin, and employed chromoprotein all has strong resistivity to heat, washing agent, reductive agent, chaotropic agent, extreme pH value and proteolytic enzyme etc., can reach the effect that silk fabrics is never faded.

(4) employed reagent and dyestuff mostly are acid or alkali in the silk dyeing and finishing technology, and these chemical substances not only can influence the quality of silk, health that also can harm humans.The chromatic cocoon that the present invention created then can be crossed this step of dyeing and finishing, has not only guaranteed the quality of silk, and provides guarantee for human beings'health.

(5) chromoprotein that uses among the present invention all belongs to GFP family basically, and the absorption spectrum of the fluorescence of GFP family and the chromoprotein of non-fluorescence has all covered UV-light zone and based on shortwaves such as ultraviolets.Thereby can absorb ultraviolet ray with the fabric that this material is made and reduce the injury of ultraviolet ray the mankind.

(6) chromoprotein can absorb ultraviolet ray efficiently and be translated into the light or the heat energy of another kind of long-wave band, and the fabric made from this material may have unique optical characteristics and purposes widely.

Method used in the present invention is disposable to have solved that tame silk cocoon look color category is few, pigment is in conjunction with unstable two big important difficult problems, that can simplify silk fabrics spins clothes technology, eliminate the harm of dyeing and finishing technology, will increase new good speciality for the silk of the good reputation of just enjoying " fiber queen " originally to silk quality and human health.

Embodiment:

Embodiment 1:

Utilize practical cultivated silkworm breed variety " bluish waves " (white cocoon kind) as starting materials, the oolysis just given birth to is removed after the diapause in giving birth to back 3-6h, use the Eppendorf microinjection instrument, with 10-15nl, the red fluorescent protein mark that has neural and the startup of eyes specific promoter of total concn 400ng/ul and the reorganization piggyBac carrier and the assistant carrier of the green fluorescent protein (green fluorescence chromoprotein) that the fibroin heavy chain promoter starts, common injection enters in 1500 silkworm seeds, seal injection port with nontoxic glue, in 35% formaldehyde vapors the sterilization 5min after, place 25 ℃, the hatching of hastening the hatching of silkworms in the high humidity environment of relative humidity more than 85%, 73 G0 of hatching are collected raising to changing moth for newly-hatched silkworm with artificial diet, 40 G0 that obtain are for silkworm moth, enclose G1 for silkworm seed by the selfing or common acquisition 27 moths of backcrossing, use

Electronic macroscopical fluorescence microscope screens 27 moths circles G1 for silkworm seed and obtain 3 positive moths circles, totally 18 transgenic silkworms.18 transgenic silkworms that obtain are raised to the harvesting of cocoon of putting on an arrow, obtain 18 absinthe-green silk cocoons that present in various degree altogether.

Embodiment 2:

Utilize practical cultivated silkworm breed variety " Dongting Lake " (white cocoon kind) as starting materials, the oolysis just given birth to is removed after the diapause in giving birth to back 3-6h, use the Eppendorf microinjection instrument, with 10-15nl, the red fluorescent protein mark that has neural and the startup of eyes specific promoter of total concn 400ng/ul and the reorganization piggyBac carrier and the assistant carrier of the blue fluorescent protein (blue-fluorescence chromoprotein) that the fibroin heavy chain promoter starts, common injection enters in 1800 silkworm seeds, seal injection port with nontoxic glue, in 36% formaldehyde vapors the sterilization 5min after, place 25 ℃, the hatching of hastening the hatching of silkworms in the high humidity environment of relative humidity more than 85%, 150 G0 of hatching are collected raising to changing moth for newly-hatched silkworm with artificial diet, 100 G0 that obtain are for silkworm moth, enclose G1 for silkworm seed by the selfing or common acquisition 62 moths of backcrossing, use

Electronic macroscopical fluorescence microscope screens 62 moths circles G1 for silkworm seed and obtain 15 positive moths circles, totally 120 transgenic silkworms.120 transgenic silkworms that obtain are raised to the harvesting of cocoon of putting on an arrow, obtain 113 silk cocoons that present blueness in various degree altogether.

Embodiment 3:

Utilize practical cultivated silkworm breed variety " 872 " (white cocoon kind) as starting materials, the oolysis just given birth to is removed after the diapause in giving birth to back 3-6h, use the Eppendorf microinjection instrument, with 10-15nl, the red fluorescent protein mark that has neural and the startup of eyes specific promoter of total concn 400ng/ul and the reorganization piggyBac carrier and the assistant carrier of the yellow fluorescence protein (yellow fluorescence chromoprotein) that the fibroin heavy chain promoter starts, common injection enters in 2000 silkworm seeds, seal injection port with nontoxic glue, in 37% formaldehyde vapors the sterilization 5min after, place 25 ℃, the hatching of hastening the hatching of silkworms in the high humidity environment of relative humidity more than 85%, 142 G0 of hatching are collected raising to changing moth for newly-hatched silkworm with artificial diet, 99 G0 that obtain are for silkworm moth, enclose G1 for silkworm seed by the selfing or common acquisition 45 moths of backcrossing, use

Electronic macroscopical fluorescence microscope screens 45 moths circles G1 for silkworm seed and obtain 12 positive moths circles, totally 80 transgenic silkworms.80 transgenic silkworms that obtain are raised to the harvesting of cocoon of putting on an arrow, obtain 69 xanchromatic silk cocoons that present in various degree altogether.

Embodiment 4:

Utilize practical cultivated silkworm breed variety " 871 " (white cocoon kind) as starting materials, the oolysis just given birth to is removed after the diapause in giving birth to back 3-6h, use the Eppendorf microinjection instrument, with 10-15nl, the red fluorescent protein mark that has neural and the startup of eyes specific promoter of total concn 400ng/ul and the reorganization piggyBac carrier and the assistant carrier of the carmetta chromoprotein (carmetta non-fluorescent chromoprotein) that the fibroin heavy chain promoter starts, common injection enters in 2500 silkworm seeds, seal injection port with nontoxic glue, in 35% formaldehyde vapors the sterilization 5min after, place 25 ℃, the hatching of hastening the hatching of silkworms in the high humidity environment of relative humidity more than 85%, 200 G0 of hatching are collected raising to changing moth for newly-hatched silkworm with artificial diet, 180 G0 that obtain are for silkworm moth, enclose G1 for silkworm seed by the selfing or common acquisition 87 moths of backcrossing, use

Electronic macroscopical fluorescence microscope screens 87 moths circles G1 for silkworm seed and obtain 20 positive moths circles, totally 150 transgenic silkworms.150 transgenic silkworms that obtain are raised to the harvesting of cocoon of putting on an arrow, obtain 134 silk cocoons that present redness in various degree altogether.

Embodiment 5:

Utilize practical cultivated silkworm breed variety " autumn is white " (white cocoon kind) as starting materials, the oolysis just given birth to is removed after the diapause in giving birth to back 3-6h, use the Eppendorf microinjection instrument, with 10-15nl, the red fluorescent protein mark that has neural and the startup of eyes specific promoter of total concn 400ng/ul and the reorganization piggyBac carrier and the assistant carrier of the blue pigment albumen (blue non-fluorescent chromoprotein) that the fibroin heavy chain promoter starts, common injection enters in 2300 silkworm seeds, seal injection port with nontoxic glue, in 35% formaldehyde vapors the sterilization 5min after, place 25 ℃, the hatching of hastening the hatching of silkworms in the high humidity environment of relative humidity more than 85%, 106 G0 of hatching are collected raising to changing moth for newly-hatched silkworm with artificial diet, 89 G0 that obtain are for silkworm moth, enclose G1 for silkworm seed by the selfing or common acquisition 53 moths of backcrossing, use

Electronic macroscopical fluorescence microscope screens 53 moths circles G1 for silkworm seed and obtain 8 positive moths circles, totally 82 transgenic silkworms.82 transgenic silkworms that obtain are raised to the harvesting of cocoon of putting on an arrow, obtain 75 silk cocoons that present blueness in various degree altogether.

Embodiment 6:

Utilize practical cultivated silkworm breed variety " bluish waves " (white cocoon kind) as starting materials, the oolysis just given birth to is removed after the diapause in giving birth to back 3-6h, use the Eppendorf microinjection instrument, with 10-15nl, the red fluorescent protein mark that has neural and the startup of eyes specific promoter of total concn 400ng/ul and the reorganization piggyBac carrier and the assistant carrier of the scarlet chromoprotein that the fibroin heavy chain promoter starts, common injection enters in 2100 silkworm seeds, seal injection port with nontoxic glue, in 36% formaldehyde vapors the sterilization 5min after, place 25 ℃, the hatching of hastening the hatching of silkworms in the high humidity environment of relative humidity more than 85%, 96 G0 of hatching are collected raising to changing moth for newly-hatched silkworm with artificial diet, 81 G0 that obtain are for silkworm moth, enclose G1 for silkworm seed by the selfing or common acquisition 43 moths of backcrossing, use Electronic macroscopical fluorescence microscope screens 53 moths circles G1 for silkworm seed and obtain 3 positive moths circles, totally 24 transgenic silkworms.24 transgenic silkworms that obtain are raised to the harvesting of cocoon of putting on an arrow, obtain 24 wine-colored silk cocoons that present in various degree altogether.

Claims (1)

1, utilize the method for chromoprotein cultivated silkworm chromatic cocoon, it is characterized in that realizing by following steps successively:

(1) obtaining of chromoprotein gene: the respective organization of choosing karang or other relevant marine organism is extracted genome or synthetic relevant cDNA, amplify the gene fragment of expressing chromoprotein, perhaps directly buy the carrier that contains the chromoprotein gene to relevant biotech firm;

(2) structure of silkworm transgene carrier: according to the requirement of the public transgenic technology of silkworm at present, make up the piggyBac expression vector, the red fluorescent protein (DsRed) that starts with the promotor 3xp3 of eye and neural specific starts the chromoprotein gene that obtains with the bombyx mori silk fibroin heavy chain promoter and realizes amalgamation and expression as selection markers;

(3) silkworm embryos microinjection: with white cocoon cultivated silkworm breed variety as starting materials, the oolysis just given birth to removed after the diapause at the back 3h-6h of laying eggs reorganization transposon vector plasmid and the assistant carrier plasmid of 10-15nl, total concn 400ng/ul injected the silkworm seed of termination of diapause from silkworm seed outside of belly central authorities, and inject the aperture that stays with nontoxic glue sealing chorion with microinjection instrument;

(4) screening of transgenic silkworm: the silkworm seed that injection is finished is sterilized in the formaldehyde vapors of 35-37% behind the 5min, place under 25 ℃, the condition of relative humidity more than 85%, hasten the hatching of silkworms up to hatching, the newly-hatched silkworm that raising hatches out, the silkworm moth in the present age (G0) is carried out selfing or backcrosses, obtain G1 for silkworm seed, scanning G1 obtains the transgenosis individuality for silkworm seed, promptly obtains the transgenosis individuality of respective color silk cocoon;

(5) acquisition of colored silk cocoon transgenic strain material: with the transgenic positive individual feeding that screens to put on an arrow harvesting of cocoon and further selfing select pure, promptly obtain can genetic stability colored silk cocoon transgenic strain material.

In the above-mentioned steps, h represents hour, and min represents minute.