CN101948848A - Application of corn anthocyanin regulatory gene Lc in quick-cultivation red rice paddy with high flavone content, method and expression vector - Google Patents
Application of corn anthocyanin regulatory gene Lc in quick-cultivation red rice paddy with high flavone content, method and expression vector Download PDFInfo
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Abstract
The invention relates to the technical field of plant genetic engineering, in particular to the application of a corn anthocyanin regulatory gene Lc in quick-cultivation red rice paddy with high flavone content. An expression vector is constructed, wherein the expression vector comprises a promoter, a 5'UTR sequence, a corn anthocyanin regulatory gene Lc and a terminator sequentially. The expression vector is used for transforming white rice paddy and the red rice paddy with the high flavone content is obtained by screening. The construction of the vector is simpler; the red rice paddy with the high flavone content can be cultivated only by introducing one gene; and the germination percentage of transgenic descendant seeds can completely meet the requirements on paddy production.
Description
Technical field
The present invention relates to the plant gene engineering technology field, specifically, relate to corn anthocyanin regulatory gene
LcBe applied to quickly breeding high flavones content red rice paddy rice and method and expression vector.
Background technology
Paddy rice is one of most important food crop in the world, and it is staple food grain with rice that the whole world has half population at least.Along with growth in the living standard, the functional rice rearing new variety with higher health care value more and more is subjected to people's attention.
Flavonoid claims Flavonoid substances again, is the low-molecular-weight Polyphenols secondary metabolite that a class extensively exists in vegitabilia.At present existing more bibliographical information, flavonoid compound is the very strong material of a class biological activity, it is a kind of natural antioxidant, have effects such as the myocardial consumption of oxygen of reduction, treatment of vascular sclerosis, have effect (Knekt etc. such as anti-ageing, enhancing body immunizing power, anti-cancer and cancer-preventing, Am. J. Epidemiol., 1997,146 (3): 223-230; Knekt etc., Eur. J. Clin. Nutr., 2000,54 (5): 415-417; Middleton etc., Pharmacol Rev., 2000,52 (4): 673-751; Williams etc., Free Radical Biol Med., 2004,36 (7): 838 – 849).Flavonoid substances has various health care functions to human body, and it more and more is subject to people's attention in the effect that strengthens body immunity and prevent and treat aspects such as multiple chronic disease at present.
Contain the important member in the flavonoid in coloured rice, cyanidin(e) combines the anthocyanogen that forms with glycosyl.Tests such as Koide show that the anthocyanin hydrolysate of red rice can obviously suppress growth (Koide etc., Cancer Biother Radiopharm, 1996,11 (4): 273-277) of tumour cell.Ling etc. studies show that, adopt the red rice fr raising rabbits, can reduce the atherosclerotic plaque area, but also can improve serum high-density LP cholesterol (HDL) and apolipoprotein AI (ApoAI) level, liver homogenate active oxygen (ROS) and aorta mda (MDA) index also significantly reduce simultaneously.They think that the reason that produces this phenomenon may be to promote the contrary of cholesterol to turn round by improving HDL and ApoAI level, thereby but the removing of acceleration bodies inner cholesterol reaches antiatherogenic effect, this effect may have relevant than high-micro-element with coloured rice, also may be because the ROS level reduces the oxidation resistant ability (Ling etc. that improved body in the body, J.Nutr, 2001,131 (5): 1421-1426).
Institute of Crop Science, Chinese Academy of Agricultural Science Han Longzhi researcher laboratory, in " the genetic research progress of paddy rice anthocyanin content " summary one literary composition of delivering in 2006, also summarized at present both at home and abroad about the nourishing function of coloured paddy rice human body, growth (the Sun Mingmao etc. that can obviously suppress tumour cell as red rice rice anthocyanogen hydrolyzate, the plant genetic resources journal, 2006,7 (2): 239-245).The Diplomarbeit that professor Xu Shibo of biology department of Zhongshan University instructs Master degree candidate He Rongfei to finish, studied of the pharmacological action of paddy rice total flavones to senile dementia, and point out that the paddy rice flavones is to the experimental mouse senile dementia symptom effect (He Rongfei etc. that have clear improvement, Chinese medicinal materials, 2002,25(2): 108-111); Xu Donghui etc. reported the paddy rice flavones can obviously alleviate rat experiment hepatic fibrosis phenomenon (Xu Donghui etc., Harbin University of Commerce's journal (natural science edition), 2002,18(1): 34-36).Coloured paddy rice also has higher nutritive value except having as above these special efficacies.Han Longzhi etc. had once analyzed them and have cultivated and obtain to have the rice nutritive ingredient of specialized character.Found that sweet red rice brown rice protein, Methionin, fat, oleic acid and linolic acid, vitamins B l and calcium contents are higher; The protein of red rice, zinc and selenium content higher (Han Longzhi etc., plant genetic resources journal, 2003,4 (3): 207-213).
Therefore, consider that as can managing to cultivate the red rice paddy rice, thereby the content that improves rice flavonoid material and other important nutritive ingredients all is extremely important from helping the human health aspect.
Natural red rice paddy rice generally belongs to wild-rice, and many proterties of wild-rice are not suitable for existing in cultivated rice, and therefore, natural red rice paddy rice can not directly be employed in Rice Production.Though present existing report (Ye Xiaoying etc. that utilize the conventional hybridization method successfully to cultivate the red rice new rice variety that can use as cultivated rice of people, Sichuan University's journal (natural science edition), 2008,45(3): 656-662), but it is well-known, adopt the required time of hybridization technique cultivation new rice variety longer, as wild red rice paddy rice and high yield conventional rice are hybridized, the F1 plant selfing of cultivating is obtained to have the F2 seed of multiple proterties combination, again by the F2 plant being filtered out multiple proterties red rice paddy rice individual plant relatively preferably, finish this process need at least three season rice cultivation, utilizing Hainan to carry out increasing winter a breeding generation also needs time a year and a half.Make screened to individual plant genetic composition isozygoty fully and may also need through the plantation screening of 6 to 7 generations.Therefore, use the conventional hybridization technology to cultivate new rice variety, utilize south numerous added-generation to accelerate the breeding progress exactly, 1 year two season of kind generally at least also needed the time in 4 years half to 5 year; And employing crossbreeding technology, because two parents respectively are 50% to the contribution rate of descendant inheritting material, if the various proterties of the rice varieties of former red rice before this are undesirable, even how to enlarge the F2 plant colony that plantation has multiple proterties combination again, also in the new rice variety of cultivating, eliminate some proterties that original unfavorable parent paddy rice stays unavoidably fully.
From the seventies in 20th century along with the foundation of genetic engineering technique and perfect, make and utilize transgenic technology improvement crop quality to become possibility.Adopt transgenic method improvement crop quality, be converted into the whole process of the offspring of isozygotying that screens from expression vector establishment, rice genetic, same 1 year two seasons of kind only needed for 2 years.And by the new rice variety that transgenic technology is cultivated, except objective trait, other proterties of plant generally can not be changed.
The flavonoid biosynthetic pathway is mainly controlled by two genoids: structure gene and regulatory gene.Wherein, the various enzymes that the structure gene direct coding is relevant with the flavonoid biosynthesizing, regulatory gene then is a genoid of control texture genetic expression intensity and phraseology.At present and the biosynthetic primary structure gene of flavonoid and regulatory gene in some plants, cloned, the biochemical action mechanism of the enzyme of primary structure genes encoding is also illustrated (Holton etc., Plant Cell, 1995,7 (7): 1071-1083; Winkel-Shirley, Plant Physiol., 2001a, 126 (2): 485-493; Winkel-Shirley, Plant Physiol., 2001b, 127 (4): 1399-1404).At present, separated, the evaluation regulatory gene relevant with the biosynthesizing of regulation and control flavonoid mainly contains two big class transcription factor: MYB and MYC(bHLH).
Corn
C1The albumen of coded by said gene is first MYB class transcription factor (Paz-Ares etc., EMBO J., 1987,6 (12): 3553-3558) of finding from plant.Corn
LcGene is one and participates in the regulatory gene (Dooner etc. that tissues such as regulation and control vein, the tip of a leaf, auricle produce anthocyanin, Genetics, 1976,82 (2): 309-322), be the 1st the transcription factor of in plant, finding (Ludwig etc., Proc. Natl. Acad. Sci. USA, 1989 with bHLH structural domain, 86 (18): 7092-7096), belong to
RThe gene family member.
People such as Llody 1992 are in the U.S. scientific magazine reported first, with corn anthocyanin regulatory gene
LcDuring heterogenous expression, the corolla eaves and the filigree of transgene tobacco become redness in tobacco and Arabidopis thaliana two kind of plant, change
LcThe Arabidopis thaliana of gene cyanidin(e) content in leaf, stem, sepal increases, but does not have the generation of cyanidin(e) at root, petal, these positions of stamen.Change over to separately
C1The Arabidopis thaliana of gene and tobacco all do not have tangible proterties.To change over to respectively
LcGene and
C1The F1 generation that the hybridization of the Arabidopis thaliana of gene obtains, can do not produce originally cyanidin(e) position such as root, petal, stamen produce cyanidin(e) (Lloyd etc.,
Science, 1992,258:1773-1775).
After, there is the scholar successively will again
LcGene difference leading-in petunia (Quattrocchio etc.,
Plant Cell, 1993,5 (11): 1497-1512; Bradley etc.,
PlantJ., 1998,13 (3): 381-392) and clover (Heather etc.,
Plant Physiol, 2003,132 (3): 1448 – 1463) in, find that also therefore the callus of some transfer-gen plant or pattern or leaf color change.Bovy etc. will
LcGene and
C1Gene together imports tomato, and the transgenic Fructus Lycopersici esculenti blade is a purple, and flavonol content improves in pulp, and total flavones alcohol (flavonols) content is 20 times of (Bovy etc., Plant Cell, 2002,14 (10): 2509-2526) of contrast.Li in 2005 etc. will
LcGene imports the double-colored bulb of fritillary of foliage plants, also caused leaf color obvious change (Li etc.,
Plant Cell Rep, 2005,23:716 – 720).People such as Li will
LcGene successfully imports apple, transgenic calli, leaf, stem all become redness, purplish red (Li etc., Planta, 2007,226:1243-1254).2005, by the Diplomarbeit that genetics specialty associate professor Zhu Dengyun of Biology College, Chinese Agriculture Univ. instructs poplar wing spring Master degree candidate to write, part work also was to utilize corn anthocyanin regulatory gene
LcTransformation of tobacco, the result shows that also change (poplar wing spring, the genetics specialty master of Biology College, Chinese Agriculture Univ. Diplomarbeit, 2005) has taken place the transgene tobacco pattern.We once guided constructive expression's CaMV35S promotor in the laboratory
LcThe gene transformation paddy rice, however the phenomenon of change all appears in the callus of transgenic paddy rice and the color of blade.
Reports such as Dan Libo are with CaMV35S promotor guiding corn
C1With
RRegulatory gene expression vector transformed wheat, corn, paddy rice and tobacco found that red color visible spot in the callus, and they think thus
C1With
RRegulatory gene can be used as index (Dan Libo, Acta Genetica Sinica, 2000,27 (1): 65-69) of weighing the particle gun changing effect.Gandikota etc. are also once with the regulatory gene of corn
C1With
R, and structure gene
C2(chalcone synthase) be rice transformation together, and red-purple appears in the rice callus tissue as a result, but other expression
C1With
R, only express
C1, or only express
RAnalogue (Gandikota etc., Molecular Breeding, 2001,7 (1): 73-83) can not appear.
2006, Shin etc. utilized the alcohol soluble protein gene promotor of rice endosperm specific expression
NPR33Guiding comes from two kinds of regulatory gene of corn
C1With
R-
SGene (
R-
SGene also is
RThe gene family member) expression vector of Gou Jianing, at rice paddy seed aleurone layer specifically expressing
C1With
R-
SGene, make common rice paddy rice brown rice become reddish-brown, flavonoid material comparison illumination shows raising after testing simultaneously, but tangible decline has appearred in grain weight, the grain of rice length and width of transgenosis brown rice, economical character such as thick, 58% and the 36%(Shin etc. that have only contrast as the transgenic paddy rice seed weight of summer and winter planting's results respectively, Plant Biotechnology, 2006,4(3): 303-315).Show according to people's results of study such as Chen Huizhe, the poor more percentage of germination of seed plumpness is low more, because reducing, plumpness cause thousand seed weight when 23.5 grams are reduced to 16.1 grams (be equivalent to have only full seed weight 68.5%) when seed, rate of emergence has only 34.6%, and the seed seedling rate of plumpness difference is also low, and the plumpness difference also can cause plant tillering power to weaken (Chen Huizhe etc. simultaneously, the Fujian agriculture science, 2004,19(2): 65-67), this can directly have influence on the output of paddy rice.In Rice Production, the conventional rice rate of emergence that requires to be used must surpass 85%(standard GB 4404.1-2008).Therefore, the transgenosis red rice by cultivations such as Shin is difficult to be employed in Rice Production.
Also there is not at present the every economical character comprehensive evaluation of a kind of quickly breeding rice high flavones content while plant method of red rice paddy rice preferably.
Summarize present people for
LcThe research of gene order (GenBank:M26227.1), in dicotyledons, people are main to be used separately
LcGene or use simultaneously
LcGene and
C1Gene is used to change pattern, or the plant color, or the color of fruit etc.In monocotyledons, except this laboratory, yet there are no independent use
LcThe monocotyledonous report of gene transformation has only at the same time and uses
C1Gene and with
LcGene is with family
RThe report of the common rice transformation of gene.
Therefore, need set up the novel method of the red rice paddy rice that a kind of quickly breeding rice flavones content is improved significantly, need breeding time oversize to overcome present employing conventional hybridization technology cultivation red rice paddy rice, being difficult to simultaneously to remove fully the output that bad parent stays in the red rice new rice variety of cultivating may be lower, unfavorable proterties such as inferior quality, or avoid simultaneously expression vector that MYB and MYC two class transcription factors are made up as goal gene rice transformation simultaneously, the workload of expression vector establishment is increased, the shrivelled bad economical character such as serious of transgenic progeny rice is followed appearance, is difficult to be employed aborning thereby have influence on the red rice paddy rice of successfully cultivating.
Summary of the invention
The objective of the invention is to be intended to corn anthocyanin regulatory gene (
LcGene) is applied to quickly breeding high flavones content red rice paddy rice.
The present invention also provides the expression vector of quickly breeding high flavones content red rice paddy rice.
The present invention also provides the method for above-mentioned quickly breeding high flavones content red rice paddy rice.
Technical scheme of the present invention is, corn anthocyanin regulatory gene (
LcGene) is applied to quickly breeding high flavones content red rice paddy rice.
The expression vector of a kind of quickly breeding high flavones content red rice paddy rice comprises following sequence successively in the sequence of plasmid vector:
A: promotor, be selected from the promotor of storage protein plasmagene in the paddy rice, be preferably gluten
GtThe promotor of 1 gene;
B:5 ' UTR sequence, 5 ' UTR sequence for same genetic unit under the promotor is preferably gluten
Gt5 ' UTR sequence of 1 gene;
C: corn anthocyanin regulatory gene
Lc
D: terminator;
5 ' UTR and goal gene in the promotor downstream
LcBetween the sequence, can also comprise the nucleotide sequence of the affiliated same genetic unit coded signal peptide fragment of promotor, promptly
GtPromotor downstream 5 ' the UTR sequence and the corn anthocyanin regulatory gene of 1 gene
LcBetween have
GtThe segmental nucleotide sequence of 1 genes encoding signal peptide.
Preferably, also comprise in the expression vector hygromycin gene and
GusReporter gene.
Above-mentioned expression vector is used to transform the rice paddy rice, obtains high flavones content red rice paddy rice through screening.
The method of a kind of quickly breeding high flavones content red rice paddy rice, step comprises:
(1) will contain corn anthocyanin regulatory gene
LcExpression vector import rice paddy rice receptor tissue, method is preferably: will contain corn anthocyanin regulatory gene
LcThe agrobacterium tumefaciens and the rice paddy rice receptor tissue of expression vector cultivate altogether; Or utilize particle bombardment with containing corn anthocyanin regulatory gene
LcExpression vector transform rice paddy rice receptor tissue;
Comprise promotor, 5 ' UTR sequence, corn anthocyanin regulatory gene in the expression vector successively
LcAnd terminator, also comprise hygromycin gene and
GusReporter gene;
Promotor is selected from the promotor of storage protein plasmagene in the paddy rice, and 5 ' UTR sequence is a 5 ' UTR sequence of same genetic unit under the promotor;
5 ' UTR and goal gene in the promotor downstream
LcBetween the sequence, can also comprise the nucleotide sequence of the affiliated same genetic unit coded signal peptide fragment of promotor;
Preferred promotor is a gluten
GtThe promotor of 1 gene, 5 ' UTR sequence is a gluten
Gt5 ' UTR sequence of 1 gene;
(2) with the paddy rice receptor tissue after the hygromycin selection conversion, regenerating and planting obtains T
0For plant;
Screening T
0Whether contain for plant
LcThe method of gene is preferably, and carries out PCR testing goal gene with the following primer shown in SEQ ID No.1 and the SEQ ID No.2:
Upstream primer LC1:5'-GCCGGCTCTCTGTCGCCGGA-3',
Downstream primer LC3:5'-GTCTGCTGCGGCCTCGCCGGTCTC-3'
From containing
LcThe T of gene
0For plant results T
1For seed, screening is the offspring of red rice;
(3) to T
1Carry out for red rice
GusReporter gene detects, and GUS is detected the red rice that endosperm is positive send out seedling, and plantation obtains T
1Plant, PCR detects and further determines T
1Whether contain for plant
LcGene;
Detection method is preferably, from T
1For getting brown rice on the red rice plant, cut the part brown rice that does not contain embryo, carry out GUS dyeing, the brown rice that endosperm is positive contains the rest part of embryo and send out seedling in substratum, and plantation obtains T
1Plant;
(4) from containing
LcThe T of gene
1For gathering in the crops T on the plant
2For seed, continue plantation T
2Obtain T for seed
2For seedling, adopt PCR testing goal gene
Lc, screening obtains not having
LcThe transgenosis of the gene isolation offspring of isozygotying.
Also can be by the following technical solutions: the method for a kind of quickly breeding high flavones content red rice paddy rice, step comprises:
(1) with expression vector B with contain corn anthocyanin regulatory gene
LcExpression vector A together transform rice paddy rice receptor tissue; Method is preferably: will contain the agrobacterium tumefaciens of expression vector A and contain the mixed of the agrobacterium tumefaciens of expression vector B according to 6～10:1, and cultivate altogether with rice paddy rice receptor tissue and transform rice paddy rice receptor tissue;
Or utilize particle bombardment, expression vector A and expression vector B according to the mixed of 6～10:1, are transformed rice paddy rice receptor tissue;
Comprise promotor, 5 ' UTR sequence, corn anthocyanin regulatory gene among the expression vector A successively
LcAnd terminator; Contain among the expression vector B hygromycin gene and
GusReporter gene;
Promotor is selected from the promotor of storage protein plasmagene in the paddy rice, and 5 ' UTR sequence is a 5 ' UTR sequence of same genetic unit under the promotor;
5 ' UTR and goal gene in the promotor downstream
LcBetween the sequence, can also comprise the nucleotide sequence of the affiliated same genetic unit coded signal peptide of promotor;
Preferred promotor is a gluten
GtThe promotor of 1 gene, 5 ' UTR sequence is a gluten
Gt5 ' UTR sequence of 1 gene;
(2) with the paddy rice receptor tissue after the hygromycin selection conversion, regenerating and planting obtains to contain
LcThe T of gene
0For plant;
Screening T
0Whether contain for plant
LcThe method of gene is preferably, and carries out PCR testing goal gene with the following primer shown in SEQ ID No.1 and the SEQ ID No.2:
Upstream primer LC1:5'-GCCGGCTCTCTGTCGCCGGA-3',
Downstream primer LC3:5'-GTCTGCTGCGGCCTCGCCGGTCTC-3'
From containing
LcThe T of gene
0For plant results T
1For seed, screening is the offspring of red rice;
(3) to T
1Carry out for red rice
GusReporter gene detects, and GUS is detected the red rice that endosperm is negative send out seedling, and plantation obtains T
1Plant; Detecting reservation through target gene PCR contains
LcThe T of gene
1For plant;
Detection method is preferably, from T
1For getting brown rice on the red rice plant, cut the part brown rice that does not contain embryo, carry out GUS dyeing, the brown rice that endosperm is negative contains the rest part of embryo and send out seedling in substratum, and plantation obtains T
1Plant;
By operation like this can not contained hygromycin gene and
GusReporter gene only contains
LcThe transfer-gen plant of gene;
(4) from containing
LcThe T of gene
1For gathering in the crops T on the plant
2For seed, continue plantation T
2Obtain T for seed
2For seedling, adopt PCR testing goal gene
Lc, screening obtains not having
LcThe transgenosis of the gene isolation offspring of isozygotying.
Further, preferred scheme is to above-mentioned two kinds of nothings that method obtains
LcThe transgenosis of the gene isolation T that isozygotys
2For in the young tender seed of bearing behind the plant blossom
LcGene carries out RT-PCR and detects, and uses paddy rice b-
ActinGene screens as internal reference
LcThe gene high expression individual plant;
Detect again
LcThe T of gene pure
2For the general flavone content of plant mature seed, keep the high transformant offspring of general flavone content.
When construction of expression vector, 5 ' UTR and goal gene in the promotor downstream
LcBetween the sequence, no matter whether insert the former affiliated segmental nucleotide sequence of genes encoding signal peptide of this promotor, can both improve the flavones content of transgenic paddy rice rice, but the nucleotide sequence that does not use the coded signal peptide improve the rice flavones content than the expression vector of the nucleotide sequence that uses the coded signal peptide can be more obvious.
Generally there is positive relationship in the raising degree of transgenic progeny brown rice shade and brown rice general flavone content, therefore, and from T
0For gathering in the crops T on the plant
1Behind seed, observe the degree that brown rice shows redness by peelling off clever shell, keep the seed of gathering in the crops on the darker individual plant of brown rice color as far as possible;
LcGenerally also there is positive relationship between gene RT-PCR detected result and the transgenic progeny brown rice general flavone content, therefore, can passes through tender seed the plant children
LcGene keeps after carrying out the RT-PCR detection
LcThe transformant of gene high expression.
Beneficial effect of the present invention is, (1) utilize crossbreeding technology can cultivate the red rice new variety, but adopt ordinary method to carry out rice breeding, operate not only that loaded down with trivial details, blindness is too big, required time is long, and some bad economical character be difficult to remove sometimes, breeding objective and result be often difficult accomplish consistent.The method of utilizing expression vector provided by the invention and cultivating transgenosis red rice paddy rice just can realize cultivating quickly and efficiently all fine high flavones content red rice paddy rice of multiple economical characters such as high yield, food flavor quality better;
(2) existing transgenic technology is carried out the red rice breeding, is with from the MYB of corn and the MYC two class transcription factors
C1With
R-
SGene while rice transformation (Shin etc., Plant Biotechnology, 2006,4(3): 303-315).Therefore, in expression vector establishment, need to make up two genetic units, i.e. two mosaic genes.The present invention only need be with in the corn MYC class transcription factor
LcThe gene transformation paddy rice can successfully be cultivated the red rice paddy rice of high flavones content.Therefore, utilize the present invention to cultivate the red rice paddy rice, the operation of construction of expression vector is more easy;
(3) utilize
C1With
R-
STwo gene while rice transformations, although also successfully cultivated the red rice paddy rice of high flavones content, but the transgenic paddy rice seed weight of gathering in the crops in Various Seasonal all obviously alleviates, the best 58%(Shin that also has only contrast etc., Plant Biotechnology, 2006,4(3): 303-315).The expression vector that uses the present invention to make up carries out rice genetic and transforms the high flavones content red rice paddy rice of cultivating, and the percentage of germination of transgenic progeny seed can satisfy the requirement on the Rice Production fully;
(4) can surpass 30 if the expression vector that the present invention is made up carries out the independent transformant that rice conversion obtains, in transgenic progeny, can obtain the brown rice general flavone content and improve offspring more than 300% than non-transgenic paddy rice brown rice.
Description of drawings
Fig. 1 is the expression vector that embodiment 1 makes up, and wherein (A) is containing by the paddy rice gluten of embodiment 1 structure
Gt1 gene promoter and 5 ' UTR sequence and contain or do not contain
GtThe nucleotide sequence guiding popcorn pigment glycosides regulatory gene of 1 genes encoding signal peptide
LcExpression vector: pCAMBIA1301-
Gt1-
Lc-Nos or pCAMBIA1301-
Gt1(S)-
LcThe T-DNA structure iron of-Nos, on the T-DNA of these two expression vectors, also have simultaneously hygromycin gene and
GusReporter gene;
Fig. 1 (B) is containing by the paddy rice gluten of embodiment 2 structures
Gt1 gene promoter and 5 ' UTR sequence and contain or do not contain
GtThe nucleotide sequence guiding popcorn pigment glycosides regulatory gene of 1 genes encoding signal peptide
LcExpression vector: pCAMBIA1300-
Gt1-
Lc-35Ster or pCAMBIA1300-
Gt1(S)-
LcThe T-DNA structure iron of-35Ster, on the T-DNA of these two expression vectors, do not have hygromycin gene and
GusReporter gene.
Fig. 2 is embodiment 3 part T
0Detect figure for transfer-gen plant and non-transgenic contrast paddy rice PCR.Among the figure: M is standard molecular weight DNA; Z is for being the product that template is carried out pcr amplification with construction of expression vector of the present invention; C is the product of template pcr amplification for contrasting oryza sativa genomic dna with non-transgenic; 1-21 is that part transfer-gen plant genomic dna is the product of template pcr amplification.
Fig. 3 is embodiment 3 part T
0For transgenic paddy rice and non-transgenic contrast paddy rice brown rice character observation.Among the figure: A is a non-transgenic contrast paddy rice; B has transformed pCAMBIA1301-from one
Gt1-
LcThe transgenosis T of-Nos expression vector T-DNA
0The red rice of gathering in the crops on the plant.
Fig. 4 is the RT-PCR detection figure of embodiment 3.Wherein Fig. 4 A is at non-transgenic contrast and part transgenosis (pCAMBIA1301-
Gt1-
L c-after Nos) paddy rice bloomed 15 days to the endogenous β of the tender seed of children-
ActinThe RT-PCR of gene cDNA quality after leveling detects figure; Fig. 4 B is at non-transgenic contrast and part transgenosis (pCAMBIA1301-
Gt1-
Lc-after Nos) paddy rice bloomed 15 days to the tender seed of children
LcGene RT-PCR detects figure.Among the figure: C is a non-transgenic contrast paddy rice; C3, C4, C6, C8, C9, C12, C14, C17, C19, C22 are and have transformed expression vector pCAMBIA1301-
Gt1-
LcThe transfer-gen plant of-Nos T-DNA.
Fig. 5 is the T-DNA structure iron of pCAMBIA1301 plasmid among the embodiment 1.Among the figure: LB, RB are the border, the left and right sides of T-DNA; 35S-pro, 35S-ter are CaMV 35S gene promoter and terminator;
HptBe hygromycin gene;
GusBe b-glucuronic acid Glycosylase gene; Nos-ter is the rouge alkali synthetase gene terminator; Xh, E, Sa, K, S, B, X, Sal, P, H are respectively
XhoI,
EcoRI,
SacI,
KpnI,
SmaI,
BamHI,
XbaI,
SalI,
PstI,
HindThe abbreviation of III, and represent corresponding restriction enzyme site.
Fig. 6 is the T-DNA structure iron of pCAMBIA1300 plasmid among the embodiment 2.Among the figure: LB, RB are the border, the left and right sides of T-DNA; 35S-pro, 35S-ter are CaMV 35S gene promoter and terminator;
HptBe hygromycin gene; Xh, E, Sa, K, S, B, X, Sal, P, H are respectively
XhoI,
EcoRI,
SacI,
KpnI,
SmaI,
BamHI,
XbaI,
SalI,
PstI,
HindThe abbreviation of III, and represent corresponding restriction enzyme site.
Embodiment
Present embodiment is to contain in order to illustrate
LcGene has hygromycin gene simultaneously and reaches
GusThe expression vector of reporter gene: pCAMBIA1301-
Gt1-
Lc-Nos(does not contain
Gt1The nucleotide sequence of genes encoding signal peptide) and pCAMBIA1301-
Gt1(S)-
Lc-Nos(contains
Gt1The nucleotide sequence of genes encoding signal peptide) structure.
Shown in Fig. 1 (A), among the figure
Gt1-pro is the paddy rice gluten
Gt1 genes encoding chain ATG upstream 5.3kb sequence comprises
Gt1 gene promoter and 5 ' UTR sequence;
Gt1-pro(S) except containing the paddy rice gluten
GtOutside the 1 genes encoding chain ATG upstream 5.3kb sequence, also contain
GtThe nucleotide sequence of 1 genes encoding signal peptide;
LcBe corn anthocyanin regulatory gene; Nos-ter is the rouge alkali synthetase gene terminator; 35S-pro, 35S-ter are respectively CaMV 35S gene promoter and terminator;
HptBe hygromycin gene;
GusBe b-glucuronic acid Glycosylase gene; LB, RB are respectively the left and right border of T-DNA; Xh, E, S, X, H, N are respectively restriction endonucleases
XhoI,
EcoRI,
SmaI,
XbaI,
HindIII,
NcoThe abbreviation of I, and represent corresponding restriction enzyme site.
Specific operation process is as follows:
A) be carrier with the pCAMBIA1301 plasmid, be called for short p1301, come from Chinese Academy of Sciences's Shanghai plant physiology and the Wang Zongyang researcher of ecological Studies institute, T-DNA structure such as Fig. 5, wherein LB, RB are the left and right border of T-DNA; 35S-pro, 35S-ter are CaMV 35S gene promoter and terminator;
HptBe hygromycin gene;
GusBe b-glucuronic acid Glycosylase gene; Nos-ter is the rouge alkali synthetase gene terminator; Xh, E, Sa, K, S, B, X, Sal, P, H, N are respectively
XhoI,
EcoRI,
SacI,
KpnI,
SmaI,
BamHI,
XbaI,
SalI,
PstI,
HindIII,
NcoThe abbreviation of I, and represent corresponding restriction enzyme site;
Design the PCR primer at pCAMBIA1301 plasmid vector Nos terminator two ends:
5 '-CCCGGGATCGTTCAAACATTTGG-3 ' and
5 '-GAATTCCCCGATCTAGTAACATAG-3 ', upstream and downstream primer 5 ' end is introduced respectively
SmaI and
EcoRThe I restriction enzyme site; The PCR product is connected with the T carrier obtains the T-1 carrier, and the transformed into escherichia coli competent cell; Use then
SmaI and
EcoRI is carried out enzyme to the T-1 plasmid and is cut, and electrophoresis reclaims small segment, with this small segment and pCAMBIA1301 plasmid
SmaI and
EcoRThe I enzyme is cut product and is connected, and obtains the pCAMBIA1301-Nos carrier, the transformed into escherichia coli competent cell; Adopt PCR, enzyme is cut and check order evaluation;
B) submit with reference to this laboratory
Gt1 gene order (GenBank:AY649098),
Gt5319bp place, 1 gene coding region initiator codon ATG upstream design upstream primer: 5 '-GGAAGCTTTCTCCGGGTCATCGATAGGTGGGGC-3 ',
Gt1 gene 5 ' UTR downstream end design downstream primer: 5'-GGGTCTAGAGTTGTTGTAGGACTAATGAACTGA-3' or
Gt72 nucleotides downstream end design downstream primers of 1 genes encoding signal peptide: 5 '-GGGTCTAGAGGCTAGGGAGCCATCGCACAAG-3 ', upstream and downstream primer 5 ' end is introduced respectively
HindIII and
XbaThe I restriction enzyme site is that template is carried out the pcr amplification acquisition with the conventional rice leaf DNA
GtThe nucleotide sequence of 1 gene 5.3kb promotor and 5 ' UTR or 5.3kb promotor and 5 ' UTR and coded signal peptide is connected the PCR product and obtains the T-2 carrier with the T carrier, and the transformed into escherichia coli competent cell; By enzyme cut, PCR and order-checking identify the T-2 carrier; Use then
HindIII and
XbaI is carried out enzyme to the T-2 plasmid and is cut, and electrophoresis reclaims big fragment, should big fragment and pCAMBIA1301-Nos plasmid
HindIII and
XbaThe I enzyme is cut product and is connected, and obtains pCAMBIA1301-
Gt1-Nos or pCAMBIA1301-
Gt1(S)-and the Nos carrier, the transformed into escherichia coli competent cell; Adopt PCR, enzyme is cut and check order evaluation;
C) announce with reference to GenBank
LcGene order (GenBank:M26227.1),
LcGenes encoding chain initiating codon ATG and terminator codon TGA place design the upstream and downstream primer respectively: 5 '-GTCTAGAATGGCGCTTTCAGCTTGG-3 ' and 5 '-CCCGGGTCACCGCTTCCCTATAG-3 ', primer 5 ' end is introduced respectively
XbaI and
SmaThe I restriction enzyme site.Extracting maize leaf RNA obtains by reverse transcription again
LcGene coded sequence is connected the RT-PCR product and obtains the T-3 carrier with the T carrier, and the transformed into escherichia coli competent cell; By enzyme cut, PCR and order-checking identify the T-3 carrier; Use then
SmaI and
XbaI is carried out enzyme to the T-3 plasmid and is cut, and electrophoresis reclaims small segment, with this small segment and pCAMBIA1301-
Gt1-Nos or pCAMBIA1301-
Gt1(S)-Nos carrier warp
SmaI and
XbaThe I enzyme is cut product and is connected, and obtains pCAMBIA1301-
Gt1-
Lc-Nos and pCAMBIA1301-
Gt1(S)-
Lc-Nos carrier, the transformed into escherichia coli competent cell; Adopt PCR, enzyme is cut and check order evaluation.
What make up contains by the paddy rice gluten
Gt1 gene promoter and 5 ' UTR sequence and contain or do not contain
GtThe nucleotide sequence guiding popcorn pigment glycosides regulatory gene of 1 genes encoding signal peptide
LcThe T-DNA structure iron of expression vector shown in Fig. 1 (A), also have simultaneously hygromycin gene and
GusReporter gene.
Present embodiment is to contain in order to illustrate
LcGene does not have hygromycin gene and reaches
GusThe expression vector of reporter gene: pCAMBIA1300-
Gt1-
Lc-35Ster(does not contain
Gt1The nucleotide sequence of genes encoding signal peptide) and pCAMBIA1300-
Gt1(S)-
Lc-35Ster (contains
Gt1The nucleotide sequence of genes encoding signal peptide) structure, shown in Fig. 1 (B), on the T-DNA of these two expression vectors, do not have hygromycin gene and
GusReporter gene.
Specific operation process is as follows:
A) be carrier with the pCAMBIA1300 plasmid, be called for short p1300, come from Chinese Academy of Sciences's Shanghai plant physiology and the Wang Zongyang researcher of ecological Studies institute, the T-DNA structure iron as shown in Figure 6.Wherein LB, RB are the border, the left and right sides of T-DNA; 35S-pro, 35S-ter are CaMV 35S gene promoter and terminator;
HptBe hygromycin gene; Xh, E, Sa, K, S, B, X, Sal, P, H are respectively
XhoI,
EcoRI,
SacI,
KpnI,
SmaI,
BamHI,
XbaI,
SalI,
PstI,
HindThe abbreviation of III, and represent corresponding restriction enzyme site;
B) submit with reference to this laboratory equally
Gt1 gene order (GenBank:AY649098),
Gt5319bp place, 1 gene coding region initiator codon ATG upstream design upstream primer,
Gt1 gene 5 ' UTR downstream end design downstream primer:
5 '-GGAAGCTTTCTCCGGGTCATCGATAGGTGGGGC-3 ' and
5'-GGGTCTAGAGTTGTTGTAGGACTAATGAACTGA-3'，
Or
Gt72 Nucleotide end design downstream primers of 1 genes encoding signal peptide: 5 '-GGGTCTAGAGGCTAGGGAGCCATCGCACAAG-3 ', upstream and downstream primer 5 ' end is introduced respectively
HindIII and
XbaThe I restriction enzyme site is that template is carried out the pcr amplification acquisition with the conventional rice leaf DNA
GtThe nucleotide sequence of 1 gene 5.3kb promotor and 5 ' UTR or 5.3kb promotor and 5 ' UTR and coded signal peptide is connected the PCR product and obtains the T-4 carrier with the T carrier, and the transformed into escherichia coli competent cell; By enzyme cut, PCR and order-checking identify the T-4 carrier; Use then
HindIII and
XbaI is carried out enzyme to the T-4 plasmid and is cut, and electrophoresis reclaims big fragment, should big fragment and pCAMBIA1300 plasmid
HindIII and
XbaThe I enzyme is cut product and is connected, and obtains pCAMBIA1300-
Gt1 or pCAMBIA1300-
Gt1(S) carrier, the transformed into escherichia coli competent cell; Adopt PCR, enzyme is cut and check order evaluation;
C) announce with reference to GenBank equally
LcGene order (GenBank:M26227.1),
LcGenes encoding chain initiating codon ATG and terminator codon TGA design the upstream and downstream primer respectively:
5 '-GTCTAGAATGGCGCTTTCAGCTTG-3 ' and
5′-CTCGAGTCACCGCTTCCCTATAG-3′，
Primer 5 ' end is introduced respectively
XbaI and
XhoThe I restriction enzyme site.Extracting maize leaf RNA obtains by reverse transcription again
LcGene coded sequence is connected the RT-PCR product and obtains the T-5 carrier with the T carrier, and the transformed into escherichia coli competent cell; By enzyme cut, PCR and order-checking identify the T-5 carrier; Use then
XhoI and
XbaI is carried out enzyme to the T-5 plasmid and is cut, and electrophoresis reclaims small segment, with this small segment and pCAMBIA1300-
Gt1 or pCAMBIA1300-
Gt1(S) carrier warp
XhoI and
XbaThe I enzyme is cut product and is connected, and obtains pCAMBIA1300-
Gt1-
Lc-35ter and pCAMBIA1301-
Gt1(S)-
Lc-35ter carrier, the transformed into escherichia coli competent cell; Adopt PCR, enzyme is cut and check order evaluation.
Embodiment 3:
Present embodiment is for containing with embodiment 1 preparation is described
LcGene has hygromycin gene simultaneously and reaches
GusThe expression vector of reporter gene: pCAMBIA1301-
Gt1-
Lc-Nos or pCAMBIA1301-
Gt1(S)-
Lc-Nos transforms the process of common rice rice cultivating high flavones content red rice paddy rice.
(1) chooses high-yield rice new lines " super 2-10 " as utilizing transgenic technology to cultivate the acceptor material of high flavones content red rice paddy rice.At first the embryo with " super 2-10 " the paddy rice mature seed or the young tender seed of blooming 12 ~ 15 days carries out callus induction on callus inducing medium, with the callus that induces with contain embodiment 1 gained pCAMBIA1301-
Gt1-
Lc-Nos carrier or pCAMBIA1301-
Gt1(S)-
LcThe agrobacterium tumefaciens of-Nos carrier carries out common cultivation, or uses particle gun directly will contain
LcThe carrier of goal gene imports the callus of " super 2-10 " paddy rice.
(2) through the screening of two-wheeled hygromycin resistance, pre-differentiation, differentiation and root culture from the resistant calli seedling that regenerates.Obtain T through conventional the cultivation again
0Plant.
Utilize and goal gene
LcThe upstream and downstream primer of autosyndetic pairing is to transgenosis T
0Plant carries out PCR and detects, and the result shows that all transfer-gen plants can both amplify the 369 bp bands identical with expression vector, and the non-transgenic paddy rice does not have amplified band;
Upstream primer LC1:5'-GCCGGCTCTCTGTCGCCGGA-3',
Downstream primer LC3:5'-GTCTGCTGCGGCCTCGCCGGTCTC-3';
Part T
0Detect figure as shown in Figure 2 for transfer-gen plant and non-transgenic contrast paddy rice PCR, wherein M is standard molecular weight DNA; Z is for being the product that template is carried out pcr amplification with construction of expression vector of the present invention; C is the product of template pcr amplification for contrasting oryza sativa genomic dna with non-transgenic; 1-21 is that part transfer-gen plant genomic dna is the product of template pcr amplification;
From T
0Gather in the crops T on the plant
1Seed.Two types of transgenic paddy rice grain husks of strip off shell, the result shows, is transforming pCAMBIA1301-
Gt1-
LcThere is half plant brown rice to present in various degree redness among the offspring of-Nos carrier approximately; Transforming pCAMBIA1301-
Gt1(S)-
LcThere is 1/3 plant brown rice to present in various degree redness among the offspring of-Nos carrier approximately;
Part T
0Contrast as shown in Figure 3 for transgenic paddy rice and non-transgenic contrast paddy rice brown rice character observation, wherein A is a non-transgenic contrast paddy rice; B has transformed pCAMBIA1301-from one
Gt1-
LcThe transgenosis T of-Nos expression vector T-DNA
0The red rice of gathering in the crops on the plant, color is reddish-brown.Although transgenic paddy rice brown rice reduces slightly than non-transgenic contrast paddy rice,, its outward appearance is normal, shrivelled phenomenon do not occur.
(3) to being rendered as the T of red rice
1Carry out for brown rice
GusReporter gene detects, and cuts half brown rice that does not contain embryo and carries out GUS dyeing, GUS is detected half brown rice in addition that endosperm is positive send out seedling with the 1/2MS substratum, plants acquisition T
1For plant; Adopt PCR further to confirm at T
1For whether containing in the plant
LcGene.
(4) from containing
LcThe T of gene
1For gathering in the crops T on the plant
2For seed, continue plantation T
2Obtain T for seed
2For seedling, adopt PCR testing goal gene
Lc, screening obtains not having
LcThe transgenosis of the gene isolation offspring of isozygotying.
With the β of paddy rice-
ActinGene contrasts as confidential reference items, isozygotys in transgenosis and gets young tender seed after individual plant was bloomed 15 days, adopts relatively two kinds of transgenic paddy rices that the carriers conversion obtains of RT-PCR
LcGene expression dose, the result shows, transforms pCAMBIA1301-
Gt1-
LcThe transgenic paddy rice of-Nos carrier
LcGene expression dose is higher relatively.
Same β with paddy rice-
ActinGene uses RT-PCR to transform pCAMBIA1301-more as confidential reference items contrasts (Fig. 4 A)
Gt1-
LcIn the tender seed of homozygous plants children of the different transformants of-Nos expression vector
LcGene expression dose filters out
LcThe transformant offspring of gene high expression (Fig. 4 B).
Observation contains
LcThe T of gene
2For brown rice, all still take on a red color, show that thus transgenosis red rice proterties can genetic stability.
With the rutin is standard substance, detects T
2For the general flavone content of plant mature seed brown rice, the result shows that the brown rice general flavone content is improved.
The comparison of brown rice general flavone content mainly is to transform pCAMBIA1301-according to improving tangible transfer-gen plant
Gt1-
LcThe type of-Nos carrier.Wherein there is part transformant seed general flavone content to improve more than 300% than non-transgenic contrast paddy rice.
The analyzing total flavones content improves the thousand seed weight of the most tangible transformant offspring brown rice, and the result shows that the transgenic paddy rice grain heavily is more than 90% of contrast.Analyze people's result of study demonstrations such as Chen Hui wise man, because reducing, plumpness cause thousand seed weight to alleviate when being 19.6 grams (be equivalent to full seed weight 83.4%) when seed by 23.5 grams, these two kinds of seed germination rate are respectively 98.2% and 97.7%, seedling rate is respectively 72.4% and 75.1%, the not remarkable (Chen Huizhe etc. of both percentage of germination and seedling rate statistical study difference, the Fujian agriculture science, 2004,19(2): 65-67).Therefore, the transgenosis red rice paddy rice of being cultivated by the present invention is the requirement that can satisfy fully on the Rice Production from the percentage of germination angle analysis.
Present embodiment is to contain in order to illustrate
LcGene does not have hygromycin gene, and
GusThe expression vector pCAMBIA1300-of reporter gene
Gt1-
Lc-35Ster transforms the process of high flavones content that common rice rice cultivating non-resistant gene, no reporter gene meet transgenosis safety, high yield, high-quality red rice paddy rice.
" precious farming 34 " paddy rice (former numbering 99-34) is bred by Baoshan District agricultural seed stock breeding station, in September, 2003 is by the Shanghai City crop varietal approval committee, through for many years, multiple spot demonstration and promoting, this kind shows as characteristic (Zhu Yifeng etc. such as big panicle many grains per panicle, output height, rice are of fine quality, wide adaptability, Shanghai Agricultural science and technology, 2007, (4): 24)." precious farming 34 " paddy rice is obtained Shanghai City scientific and technological achievement certificate in December, 2003." precious farming 34 " rice is cooked and is had good especially mouthfeel, and the food flavor sensation is suitable with the Japanese rice of high-quality.At the beginning of 2009 12 months, obtain first Shanghai City fine quality rice in the first Shanghai City fine quality rice judgment activity of Shanghai Agricultural Technology Extension Service Centre, Shanghai City Crop Science meeting, Shanghai City seed employer's organization tissue and appraise through comparison unique gold medal (is judgment criteria with grain of rice exterior quality and two project 9 sub-projects of cooking properties and palatability).
(1) be acceptor material with " precious farming 34 " paddy rice, at first will " precious farming 34 " paddy rice mature seed or the embryo of the young tender seed of blooming 12 ~ 15 days on callus inducing medium, carry out callus induction;
Contain the expression vector pCAMBIA1300-that embodiment 2 makes up
Gt1-
LcThe agrobacterium tumefaciens of-35Ster with contain the mixed of the agrobacterium tumefaciens of pCAMBIA1301 plasmid vector by 9:1, with " precious farming 34 " the rice callus tissue cultivation altogether that induces; Or with pCAMBIA1300-
Gt1-
LcThe mixed that-35Ster plasmid vector and pCAMBIA1301 plasmid vector are pressed 9:1 utilizes particle gun to import " precious farming 34 " rice callus tissue.
(2) transform the resistance screening of back rice callus tissue, pre-differentiation, differentiation and root culture and T
0Plant target gene PCR detecting operation is identical with the step (2) of embodiment 3.
(3) obtain T
0Behind transfer-gen plant, again according to people's such as Li Jianyue report (Li Jianyue etc., Science Bulletin, 2004,49(24): 2556-2561) only contain corn
LcThe transgenic paddy rice offspring screening of gene, non-resistant gene and reporter gene.
That is, cut half brown rice that does not contain embryo and carry out GUS dyeing, GUS is detected half brown rice in addition that endosperm is negative send out seedling, plant acquisition T with the 1/2MS substratum
1For plant; Adopt PCR further to detect at T
1For whether containing in the plant
LcGene.Do not contained hygromycin gene and
GusReporter gene only contains
LcThe transfer-gen plant of gene.
To adopting
LcTarget gene PCR detects non-resistant gene and reporter gene T
2Screen in the plant
LcThe RT-PCR that the gene pure offspring carries out young tender seed equally analyzes and the analysis of mature seed brown rice general flavone content, has obtained the flavones content that can be used thus than non-transgenic contrast paddy rice raising more than 300%, high yield, high-quality red rice paddy rice in Rice Production.
Claims (10)
1. corn anthocyanin regulatory gene
LcApplication aspect quickly breeding high flavones content red rice paddy rice.
2. the expression vector of a quickly breeding high flavones content red rice paddy rice is characterized in that, comprises following sequence in the sequence of plasmid vector successively:
A: promotor is selected from the promotor of storage protein plasmagene in the paddy rice;
B:5 ' UTR sequence is 5 ' UTR sequence of same genetic unit under the promotor;
C: corn anthocyanin regulatory gene
Lc
D: terminator.
3. the expression vector of the described a kind of quickly breeding high flavones content red rice paddy rice of claim 2 is characterized in that 5 ' UTR and goal gene in the promotor downstream
LcBetween the sequence, comprise the nucleotide sequence of the affiliated same genetic unit coded signal peptide fragment of promotor.
4. the expression vector of claim 2 or 3 described a kind of quickly breeding high flavones content red rice paddy rice is characterized in that, also comprise hygromycin gene and
GusReporter gene.
5. the method for a quickly breeding high flavones content red rice paddy rice is characterized in that step comprises:
(1) will contain corn anthocyanin regulatory gene
LcExpression vector import rice paddy rice receptor tissue: will contain corn anthocyanin regulatory gene
LcThe agrobacterium tumefaciens and the rice paddy rice receptor tissue of expression vector cultivate altogether; Or utilize particle bombardment with containing corn anthocyanin regulatory gene
LcExpression vector transform rice paddy rice receptor tissue;
Comprise promotor, 5 ' UTR sequence, corn anthocyanin regulatory gene in the described expression vector successively
LcAnd terminator, also comprise hygromycin gene and
GusReporter gene;
Described promotor is selected from the promotor of storage protein plasmagene in the paddy rice, and described 5 ' UTR sequence is a 5 ' UTR sequence of same genetic unit under the promotor;
(2) the paddy rice receptor tissue after transforming with hygromycin selection regenerates and plant, and the screening acquisition contains
LcThe T of gene
0For plant;
From containing
LcThe T of gene
0For plant results T
1For seed, screening is the offspring of red rice;
(3) to T
1Carry out for red rice
GusReporter gene detects, and GUS is detected the brown rice that endosperm is positive send out seedling, and plantation obtains T
1Plant, PCR detects and further determines T
1Whether contain for plant
LcGene;
(4) from containing
LcThe T of gene
1For gathering in the crops T on the plant
2For seed, continue plantation T
2Obtain T for seed
2For seedling, adopt PCR testing goal gene
Lc, screening obtains not having
LcThe transgenosis of the gene isolation offspring of isozygotying.
6. the method for a quickly breeding high flavones content red rice paddy rice is characterized in that step comprises:
(1) with expression vector B with contain corn anthocyanin regulatory gene
LcExpression vector A import rice paddy rice receptor tissue: will contain the agrobacterium tumefaciens of expression vector A and contain the mixed of the agrobacterium tumefaciens of expression vector B, and cultivate altogether with rice paddy rice receptor tissue and transform rice paddy rice receptor tissue according to 6～10:1;
Or utilize particle bombardment, expression vector A and expression vector B according to the mixed of 6～10:1, are transformed rice paddy rice receptor tissue;
Comprise promotor, 5 ' UTR sequence, corn anthocyanin regulatory gene among the expression vector A successively
LcAnd terminator; Comprise among the expression vector B hygromycin gene and
GusReporter gene;
Described promotor is selected from the promotor of storage protein plasmagene in the paddy rice, and described 5 ' UTR sequence is a 5 ' UTR sequence of same genetic unit under the promotor;
(2) the paddy rice receptor tissue after transforming with hygromycin selection regenerates and plant, and the screening acquisition contains
LcThe T of gene
0For plant;
From containing
LcThe T of gene
0For plant results T
1For seed, screening is the offspring of red rice;
(3) to T
1Carry out for red rice
GusReporter gene detects, and GUS is detected the brown rice that endosperm is negative send out seedling, and plantation obtains T
1Plant; Detecting reservation through target gene PCR contains
LcThe T of gene
1For plant;
(4) from containing
LcThe T of gene
1For gathering in the crops T on the plant
2For seed, continue plantation T
2Obtain T for seed
2For seedling, adopt PCR testing goal gene
Lc, screening obtains not having
LcThe transgenosis of the gene isolation offspring of isozygotying.
7. the method for claim 5 or 6 described a kind of quickly breeding high flavones content red rice paddy rice is characterized in that promotor is a gluten described in the step (1)
GtThe promotor of 1 gene; Described 5 ' UTR sequence is a gluten
Gt5 ' UTR sequence of 1 gene.
8. the method for claim 5 or 6 described a kind of quickly breeding high flavones content red rice paddy rice is characterized in that, the 5 ' UTR and the goal gene in promotor downstream described in the step (1)
LcBetween the sequence, also comprise the nucleotide sequence of the affiliated same genetic unit coded signal peptide fragment of promotor.
9. the method for claim 5 or 6 described a kind of quickly breeding high flavones content red rice paddy rice is characterized in that step is screened T described in (2)
0Whether contain for plant
LcThe method of gene is to carry out PCR testing goal gene with the following primer shown in SEQ ID No.1 and the SEQ ID No.2:
Upstream primer LC1:5'-GCCGGCTCTCTGTCGCCGGA-3',
Downstream primer LC3:5'-GTCTGCTGCGGCCTCGCCGGTCTC-3'.
10. the method for claim 5 or 6 described a kind of quickly breeding high flavones content red rice paddy rice is characterized in that, to the nothing that obtains
LcThe transgenosis of the gene isolation T that isozygotys
2For in the tender seed of plant children
LcGene carries out RT-PCR and detects, screening
LcThe gene high expression individual plant;
Detect T again
2For the general flavone content of plant mature seed, keep the high transformant offspring of general flavone content.
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|---|---|---|---|
| CN 201010296308 CN101948848A (en) | 2010-09-29 | 2010-09-29 | Application of corn anthocyanin regulatory gene Lc in quick-cultivation red rice paddy with high flavone content, method and expression vector |
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| CN102649966A (en) * | 2012-05-08 | 2012-08-29 | 上海师范大学 | Method for quickly cultivating sterile rice by using corn anthocyanin regulator gene Lc |
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| CN102577950A (en) * | 2012-02-09 | 2012-07-18 | 浙江大学 | Cultivating method of indigofera pseudotinctoria mats with high flavone content |
| CN102649966A (en) * | 2012-05-08 | 2012-08-29 | 上海师范大学 | Method for quickly cultivating sterile rice by using corn anthocyanin regulator gene Lc |
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Application publication date: 20110119 |