CN104911193A - Gene sequence and application thereof - Google Patents
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Abstract
The invention provides a new gene sequence and an application thereof. The gene sequence is characterized in that am adenine deoxynucleotide is inserted between the upstream-777 and the upstream-778 of the first deoxynucleotide of the first codon of a giant embryo allele coding strand corresponding to normal embryo rice, and guanine deoxynucleotide in the downstream+125 of the first deoxynucleotide of the first codon of the coding strand mutates to adenine deoxynucleotide. The GABA content, the vitamin E content, the glutathione content and the malto-oligosaccharide content of homozygous brown rice with the gene sequence are obviously higher than those of brown rice without the gene sequence. The gene sequence is transferred to normal embryo rice to obtain homozygous giant embryo progeny, so a new species of giant embryo rice with giant embryo phenotype and with obviously improved content of various nutrients in brown rice can be obtained.
Description
Technical field
The present invention relates to a kind of gene order, particularly relate to and a kind ofly plant can be made to present large prostate characteristic and make Brown Rice contain the gene order of the multiple important nutrient such as gsh and Oligomeric maltose and the application of described gene order.
Background technology
Paddy rice is one of most important food crop of the mankind, and its rice is Chinese staple food grain.If containing abundant nutritive ingredient human body to important nourishing function in rice, as γ-aminobutyric acid, vitamin-E, gsh, Oligomeric maltose etc., edible such rice not only can have the function of health care to human body, and such rice can also be used to the raw material extracting these important nutrient.
Large embryo rice is gained the name because its brown rice has larger embryo, long-pending larger than general brown rice 2-4 times usually of the idiosome of large embryo rice.
(the Maeda etc. such as Maeda, Breeding Science, 2001,51 (3): 211-213) and (Liu Linglong etc. such as Liu Linglong, Acta Agronomica Sinica, 2005,31 (10): 1265-1270) will be bred as large embryo rice brown rice immersion process after find, in brown rice to human body have important biomolecule activity material---γ-aminobutyric acid (GABA) content significantly improves than common normal chick embryo paddy rice.(the Piao Zhongze etc. such as Piao Zhongze, China's rice, 2009,3:34-35) and (Seo etc. such as Seo, J.Korean Soc.Appl.Biol.Chem., 2011,54:700-709) report, in the large embryo rice brown rice without immersion process, GABA content is also higher than normal chick embryo paddy rice content.GABA is a kind of non-protein, functional amino.Have been reported display, GABA can anxiety (Yang Shengyuan etc., Food science, 2005,26 (9): 546-551) sleep quality (the Okada T etc. of climacteric women, are improved, Nippon Shokuhin Kagaku Kaishi, 2000,47 (8): 596-603) degeneration of brain visual cortex (Leventhal etc., Science, is delayed, 2003,300 (5620): 812-815; Hua etc., Brain Research Bulletin, 2008,75:119-125) and contribute to many important effects such as brain development (Du Jiaojiao etc., Chinese disability medical science, 2007,15 (2): 2-4).
At present, the display of more bibliographical information is also had, vitamin-E (Seo etc., J.Korean Soc.Appl.Biol.Chem., 2011,54:700-709 also containing high level in large embryo rice brown rice; Zhao Zesheng etc., Shanghai Agricultural journal, 2002,18 (supplementary issue): 5-8; Wei Zhencheng etc., plant genetic resources journal, 2005,6 (4): 386-389; Zhang etc., J.Food Biochem., 2005,29:653-661; Zhang etc., CerealResearch Communications, 2007,35:1459-1468; Park etc., Genes & Genomics, 2009,31 (4): 277-282; Jeng etc., J.Sci.Food Agric, 2012,92:1468-1474).Vitamin-E is one of most important antioxidant, has good antioxidant effect to human body, can remove the free radical in human body, delay senility.In addition, also have the display of zoology test report, vitamin-E also has obvious curative effects to hyperlipidemia prevention and therapy.The research of Giyasettin etc. finds; vitamin-E significantly can reduce total cholesterol, triglyceride level and low-density lipoprotein white level in diabetes mice serum; the blood fat of diabetes mouse is raised there are provide protection (Giyasettin etc.; J.Pineal Res; 2002,32:225-230).There are some researches prove that antioxygenation is not the bioactive unique mechanism of action of vitamin-E, vitamin-E can interact with enzyme and numerous regulatory factor thus affect the structure of cytolemma especially film fat, process (the Rimbach etc. of signal transduction in regulating cell, Proc.Nutr.Soc., 2002,61 (4): 415-425).
In addition, gross protein, total fat, mineral substance and food fibre equal size aspect, some large embryo rice brown rice is also higher than common rice brown rice (Piao Zhongze etc., Chinese rice, 2009,3:34-35; Seo etc., J.KoreanSoc.Appl.Biol.Chem., 2011,54:700-709; Zhang etc., J.Food Biochem., 2005,29:653-661; Zhang etc., Cereal Research Communications., 2007,35:1459-1468; Jeng etc., J Sci Food Agric, 2012,92:1468-1474).
The giant embryo proterties of paddy rice controls by single-gene (giant embryo gene, GE), and is recessive character (Satoh and Omura, Jpn.J.Breed, 1981,31 (3): 316-326; Koh etc., Theor Appl Genet, 1996,93 (1-2): 257-261; Qian etc., Chinese Rice Res.Newslett., 1996,4 (3): 1-2).The allelotrope ge controlling rice giant embryo proterties is located in No. 7 karyomit(e)s of paddy rice, and the distance that two RFLP mark between RZ395 and CDO497 is respectively 2.4cM and 3.4cM, and the distance between two microsatellite marker RM18 and RM10 is respectively 7.7cM and 9.6cM (Koh etc., Theor.Appl.Genet., 1996,93 (1-2): 257-261).Giant embryo gene and the chromosomal RG678 molecule marker close linkage of paddy rice No. 7 are thought in assignment of genes gene mapping research, therebetween distance is 6.2cM, and and RZ395 mark between genetic distance be 13cM (Qian etc., Chinese Rice Res.Newslett., 1996,4 (3): 1-2).In recent years, the further research of people to giant embryo gene shows, be arranged in a kind of albumen (Nagasawa etc. of giant embryo gene (Os07g0603700) the encodes cytochrome P450 family on paddy rice the 7th karyomit(e), The Plant Journal, 2013, doi:10.1111/tpj.12223; Yang etc., Molecular Plant, 6 (6): 1945-1960; Chen etc., Plant Mol.Biol.Rep., 2014, doi:10.1007/s11105-014-0731-3), this albumen is named as CYP78B5 (Chen etc., Plant Mol.Biol.Rep. at present, 2014, doi:10.1007/s11105-014-0731-3).
The allelotrope being positioned at multiple giant embryo gene be found at present on paddy rice No. 7 karyomit(e)s is refer to, as ge in the reports such as Park
-1to ge
-10and ge
t.The allelic discovery of different giant embryo gene can provide important genetic resources for various large embryo rice breeding.Although reported the allelotrope of at least 11 giant embryo genes at present, these allelotrope have been all be positioned at the sudden change on Os07g0603700 gene (NCBI ReferenceSequence:NM_001066750.1) coding strand.
In addition, also do not find at present to make rice to produce while giant embryo phenotype, content of vitamin E in rice and alpha-aminobutyric acid content all increase than non-large embryo rice rice, the new giant embryo allelotrope that glutathione content, Oligomeric maltose content also together increase.
Summary of the invention
The invention provides a kind of new gene order and application thereof, to overcome the deficiency of current paddy rice in nutritive ingredient and health-care effect.
The present invention first aspect is to provide a kind of gene order, and described gene order comprises following feature:
An adenyl-deoxyribonucleotide is inserted between upstream-777 and-778 deoxynucleotide of a) first deoxynucleotide of Os07g0603700 genes encoding chain first codon.
B) downstream+125 place of first deoxynucleotide of Os07g0603700 genes encoding chain first codon, is adenyl-deoxyribonucleotide by guanine deoxyribonucleoside acid mutation.
Gene order described in the present invention first aspect, preferably at least comprises nucleotide sequence shown in SEQ ID No.1 or the nucleotide sequence with SEQ ID No.1 sequence at least 90% homology.
The described nucleotide sequence with SEQ ID No.1 sequence at least 90% homology, is more preferably and SEQ IDNo.1 sequence at least 95%, the nucleotide sequence that is more preferably 98%, is more preferably 99% homology.
Wherein, said gene sequence of the present invention can also comprise other gene fragments not affecting said gene functional nucleotide sequence, such as comprises and can bring other proterties for plant but not affect other gene orders of said gene functional nucleotide sequence.
Gene order described in the present invention first aspect, is preferably nucleotide sequence or the nucleotide sequence with SEQ ID No.1 sequence at least 90% homology shown in SEQ ID No.1.
The described nucleotide sequence with SEQ ID No.1 sequence at least 90% homology, is more preferably and SEQ IDNo.1 sequence at least 95%, the nucleotide sequence that is more preferably 98%, is more preferably 99% homology.
The present invention second aspect is to provide the application of described gene order.Described application comprises: described gene order be implanted in Oryza (Oryza) recipient plant cell genome.
Preferably, the application be applied as giant embryo gene of gene order of the present invention.
Preferably, the multiple nutrients material be applied as in raising brown rice of gene order of the present invention, as gsh, Oligomeric maltose etc.
Preferably, described application comprises: described gene order is implanted Oryza (Oryza) recipient plant cell genome, and obtains giant embryo offspring of isozygotying.
Wherein, can be separately giant embryo gene sequence described in first aspect is implanted in Oryza (Oryza) recipient plant cell genome, also can be that described gene order is implanted in Oryza (Oryza) recipient plant cell genome together with the second gene order, described second gene order be to bring other proterties for plant but not affect other one or more gene orders of said gene functional nucleotide sequence.
Wherein, described implantation can be realized by hybridization between oryza plant, or in cell, producer variation realizes, and obtains by selfing offspring of isozygotying.
Described oryza plant can be Asian Cultivated Rice (Oryza sativa), Oryza glaberrima Steud (Oryzaglaberrima), short tongue wild-rice (Oryza barthii), particle wild-rice (Oryza granulata), open up clever wild-rice (Oryza glumaepatula), wide leaf wild-rice (Oryza latifolia), long stamen wild-rice (Oryza longistaminata), south wild-rice (Oryza meridionalis), Buddhist nun's vara wild-rice (Oryza nivara), oryza officinalis (Oryza officinalis), Oryza punctata (Oryza punctata), common wild-rice (Oryza rufipogon), the general wild-rice of horse basket (Oryza malapuzhaensis), Oryza minuta (Oryza minuta), root stock wild-rice (Oryza rhizomatis), Oryza eichingeri (Oryzaeichingeri), high―strong nurserystock (Oryza alta), large sterile lemma wild-rice (Oryza grandiglumis), Australia wild-rice (Oryza australiensis), long sterile lemma wild-rice (Oryza longiglumis), Malaysia wild-rice (Oryza ridleyi), oryza meyeriana (Oryza meyeriana), short flower pesticide wild-rice (Oryzabrachyantha), extremely short grain wild-rice (Oryza schlechteri), the kinds such as Oryza coarctata, and the improving the breed arbitrarily of above-mentioned kind.
Wherein, improve the breed described in can be hybridization, backcross in any one or a few method obtain improvement of genes kind.
Wherein, described oryza plant can be any one or a few in japonica rice (Oryza sativa japonica), long-grained nonglutinous rice (Oryza sativaindica), glutinous rice (Oryza sativa var glutinosa).
Wherein, described oryza plant can be any one or a few in paddy rice, upland rice.
Wherein, described oryza plant most preferably is Color rice (pigmented rice), as pitch-black, the purple type such as black, red-black, red, brown, yellow, green.
Third aspect of the present invention is to provide a kind of method of improvement Oryza (Oryza) plant trait, comprising: by described gene order by being implanted in described oryza plant recipient cell genome.
Described method for implantation can be hybridization, backcross in any one or a few.
Preferably, described method comprises: described gene order is implanted Oryza (Oryza) recipient plant cell genome, and obtains giant embryo offspring of isozygotying.
Wherein, can be separately gene order described in first aspect is implanted in Oryza (Oryza) recipient plant cell genome, also can be that described gene order is implanted in Oryza (Oryza) recipient plant cell genome together with the second gene order, described second gene order be to bring other proterties for plant but not affect other one or more gene orders of said gene functional nucleotide sequence.
Wherein, described implantation can be implanted to oryza plant recipient cell genome as foreign gene, or can be that producer variation realizes in oryza plant cell.
In foregoing of the present invention, described implantation realizes preferably by any one or a few in hybridization, baclccrossing techniques.
In foregoing of the present invention, described oryza plant recipient cell is preferably oryza plant seed cell.
Should be understood that, the above-mentioned all respects of the present invention and various preferred embodiment thereof, can combine by those skilled in the art are unrestricted, and various combination is also contained within context of the present invention.
The oryza plant seed that isozygotys containing gene order of the present invention, obtains larger embryo, and total content of vitamin E, γ-aminobutyric acid, gsh, Oligomeric maltose content are high, have higher nutrition and health care effect.
Accompanying drawing explanation
Fig. 1 is the giant embryo gene (SEQ ID No.3) of " super 2-10 " paddy rice in embodiment 1, giant embryo gene (SEQ ID No.1) and " Japan is fine " rice Os 07g0603700 gene order (the SEQ ID No.2) comparative result of " upper No. 5, Normal University " paddy rice;
Fig. 2 is that the rice varieties giant embryo gene expression amount containing different giant embryo gene in embodiment 1 compares;
Fig. 3-1 is that the rice varieties brown rice embryo containing different giant embryo gene in embodiment 1 compares;
Fig. 3-2 compares for " upper No. 5, Normal University ", " super 2-10 ", " Japanese fine ", " M-2-565-11-3-B (ge) " large embryo rice embryo size;
Fig. 4 is amino acid mutation kind and the site schematic diagram of each allele encodes albumen of GE.
Embodiment
Embodiment 1
Choose three kinds of paddy rice:
1) containing the paddy rice " Japan is fine " of Os07g0603700 giant embryo gene (coding strand and promotor are not all suddenlyd change);
2) do not suddenly change at Os07g0603700 giant embryo gene coding strand but between coding strand first codon first deoxynucleotide upstream-777 and-778 deoxynucleotide, insert the giant embryo gene of an adenyl-deoxyribonucleotide, namely only having promotor to have the paddy rice of the giant embryo gene of sudden change " super 2-10 ";
3) between first deoxynucleotide upstream-777 and-778 deoxynucleotide of Os07g0603700 giant embryo gene coding strand first codon, an adenyl-deoxyribonucleotide is inserted, simultaneously at first deoxynucleotide downstream+125 place of coding strand first codon, be the giant embryo gene of adenyl-deoxyribonucleotide by guanine deoxyribonucleoside acid mutation, namely promotor and coding strand have the giant embryo gene of sudden change (also referred to as " gene ge in the following content of the present invention
c") paddy rice " upper No. 5, Normal University ".
Use TPS method Trace bio-element " super 2-10 ", " upper No. 5, Normal University " two kinds of rice seedling blade STb gene.Searched " Japan is fine " rice cytochrome P 450 gene (LOC_Os07g41240) sequence announced by rice genome annotation website, and intercept the sequence that this gene C DS district upstream and downstream extends 3kb and 1kb respectively.Use Peimer5 software, from Os07g0603700 gene start codon adenyl-deoxyribonucleotide (A) (+1bp) upstream-2649bp to initiator codon adenyl-deoxyribonucleotide (A) (+1bp) downstream+2090bp, point 4 sections of designs, 4 pairs of primers (table 1).Use high-fidelity enzyme KOD, respectively pcr amplification is carried out to " super 2-10 ", " upper No. 5, Normal University " rice leaf STb gene.
Carry out rubber tapping to the object band of expection molecular weight correct (table 1) to reclaim, order-checking.Use DNAMAN software to splice two kinds of rice Os 07g0603700 gene sequencing results, and sequence is compared, together compare in contrast with the Os07g0603700 gene order of " Japan is fine " paddy rice simultaneously.
Table 1, Os07g0603700 gene PCR amplimer sequence and condition
Comparison result can find (Fig. 1, suspension points represents other sequences do not shown in gene order), with the Os07g0603700 Gene sequence comparison of " Japan is fine " paddy rice, between first deoxynucleotide upstream-777 and-778 of the giant embryo gene coding strand initiator codon of " super 2-10 " paddy rice, insert an adenyl-deoxyribonucleotide (A), and other sequences are all identical with " Japan is fine " paddy rice.With the Os07g0603700 Gene sequence comparison of " Japan is fine " paddy rice, the giant embryo gene of " upper No. 5, Normal University " paddy rice except inserting except an adenyl-deoxyribonucleotide (A) between first deoxynucleotide upstream-777 and-778 of coding strand initiator codon, at first deoxynucleotide downstream+125 place of coding strand initiator codon, also sport adenyl-deoxyribonucleotide (A) by guanine deoxyribonucleoside acid (G), namely promotor and coding strand have a mutational site, and other sequences are also all identical with " Japan is fine " paddy rice.
To get after " Japan fine " paddy rice, " super 2-10 " paddy rice and " upper No. 5, Normal University " Rice Flowering the tender seed of children of 4 days, 5 days, 6 days and 7 days respectively, remove clever shell, the expression of employing quantitative PCR analysis Os07g0603700 giant embryo gene.Found that, giant embryo gene expression amount in the tender seed of the Post flowering children of 4 to 7 days of " Japan is fine " paddy rice is all very low.Giant embryo gene promotor is had to " super 2-10 " paddy rice of sudden change, in Post flowering 4 days and the tender seed of the Post flowering children of 5 days, the expression amount of giant embryo gene is also very low, but during to Post flowering 6 days, the giant embryo gene expression amount of " super 2-10 " paddy rice increases, during to Post flowering 7 days, there is again low-down level in the giant embryo gene expression amount of " super 2-10 " paddy rice.The differential expression phenomenon of giant embryo gene in " Japan is fine " and " super 2-10 " two kinds of tender seeds of paddy rice children shows, the adenyl-deoxyribonucleotide (A) inserted between first deoxynucleotide upstream-777 and-778 of giant embryo gene coding strand initiator codon is influential for the expression of giant embryo gene.Giant embryo gene promotor and coding strand are had to " upper No. 5, Normal University " paddy rice of sudden change, in the tender seed of children of Post flowering 4 days, 5 days and 6 days, expression pattern is identical with " super 2-10 " paddy rice, but when having arrived Post flowering 7 days, the giant embryo gene expression amount of " upper No. 5, Normal University " paddy rice increases (Fig. 2) further.
With reference to Fig. 3-1, " Japan is fine " that giant embryo gene coding strand and promotor are not all suddenlyd change is although paddy rice brown rice volume is maximum, but embryo seems minimum, only has " super 2-10 " paddy rice of the giant embryo gene of promoter mutation, paddy rice more greatly for embryo ratio " Japan is fine ", and " upper No. 5, Normal University " paddy rice of the giant embryo gene of sudden change is had containing promotor and coding strand, its embryo seems maximum (white arrow pointed location is the position of embryo).
Cause " upper No. 5, Normal University " and the GE gene of " M-2-565-11-3-B (ge) " two kinds of paddy rice giant embryo phenotypes all to belong to Os07g0603700 transgenation, and all belong to new Os07g0603700 allelotrope.As shown in figure 3-2, " M-2-565-11-3-B (ge) " although the embryo of paddy rice is also significantly greater than " super 2-10 " and " Japan is fine " two kinds of paddy rice, but still is less than the embryo of " upper No. 5, Normal University " paddy rice.
Table 2, " super 2-10 " and the heavy COMPARISON OF CHARACTERS of " upper No. 5, Normal University " Brown Rice embryo
| Variety name | Super 2-10 | No. 5, upper Normal University |
| Hundred brown rice weight ± standard deviation/g | 2.18±0.01A | 1.88±0.01B |
| Hundred embryo weight ± standard deviation/g | 0.12±0.01B | 0.26±0.02A |
| Embryo and brown rice weight ratio ± standard deviation (%) | 5.50％±0.46B | 13.83％±0.84A |
Note: in table, data are mean+SD (n=3).Represent to there is pole significant difference (P<0.01) between kind with capitalizations different in a line.
Giant embryo character analysis is carried out to " upper No. 5, Normal University " and " super 2-10 " paddy rice simultaneously.Result shows, and " upper No. 5, Normal University " paddy rice average hundred embryo weight and embryo and brown rice weight ratio are respectively 0.26g and 13.83%, and all pole is significantly higher than contrast " super 2-10 " paddy rice (0.12g and 5.50%) (table 2).
Report more than ten giant embryo allelotrope, as ge-1, ge-2, ge-3, ge-4, ge-5, ge-6, ge-7, ge-8, ge-9, ge-10, ge at present
tand 10217 Characters of Giant Embryo Rices, GE allelotrope in Kita-ake Characters of Giant Embryo Rice, these allelotrope are all be positioned at the sudden change on Os07g0603700 genes encoding chain.SEQ ID No.1 gene order of the present invention belongs to the allelic gene type that coding strand and promotor have sudden change, and the embryo of the paddy rice containing SEQ ID No.1 gene order of the present invention has heavily exceeded all large embryo rices reported at present.
A kind of PROTEIN C YP78B5, CYP78B5 protein structure in giant embryo gene (Os07g0603700) encodes cytochrome P450 family has three relatively conservative structural domains (Fig. 4): the leucine rich regions of N end (it and film locate about), P450 family structure territory (comprising an oxygen binding site guarded very much to exercise single oxygenation function), another structural domain deuterohemin binding domain (be combined with Fe thus exercise enzyme catalysis function) relatively guarded.By comparing with other GE allelic mutation sites, find that the mutational site of " upper No. 5, Normal University " causes protein translation at the 41st amino acid place by premature termination, only have an imperfect leucine rich regions, P450 structural domain below and deuterohemin binding domain are lost completely.And all mutational sites reported at present, except ge-5 is except the premature termination of leucine rich regions, other GE allelotrope are all in P450 family conserved domain sequence, occur single amino acids replacement or translation termination.Show for containing the giant embryo allelotrope EMBRYO IN RICE size phenotype comparative studies of different mutational sites, in the seed of identical genetic background rice varieties, the performance of embryo size may with the CYP78B5 protein function extent of damage, and the conservative of place, mutational site gene domains is relevant, ge of the present invention
callelotrope, not only there occurs sudden change in the leucine rich regions of coding strand most upstream, and also there is sudden change at promoter region, show thus, ge
callelotrope is the excellent giant embryo allelotrope having more giant embryo potentiality.
For material, brown rice content of vitamin E is analyzed with " super 2-10 " and " upper No. 5, Normal University " two kinds of paddy rice.Found that, also containing higher content of vitamin E (2.43mg/100g) in " super 2-10 " Brown Rice, two kinds of large embryo rices (2.30mg/100g and 2.41mg/100g) (Zhang etc. were reported respectively in 2005 with Zhang etc., J.Food Biochem., 2005, 29:653-661) and in 2007 report part large embryo rice (2.4mg/100g) (Zhang etc., Cereal ResearchCommunications, 2007, content of vitamin E 35:1459-1468) is close, also significantly more than part large embryo rice (the 1.02mg/100g) (Seo etc. that Seo etc. reports, J.Korean Soc.Appl.Biol.Chem., 2011, all large embryo rices (1.53IU/100g 54:700-709) reported with Wei Zhencheng etc., 1.50IU/100g and 1.40IU/100g, calculate as be equivalent to 0.668mg according to vitamin-E international unit (IU), the content of vitamin E of three kinds of large embryo rices of the report such as Wei Zhencheng is respectively 1.02204mg/100g, 1.002mg/100g and 0.9352mg/100g) (Wei Zhencheng etc., plant genetic resources journal, 2005, 6 (4): 386-389) content of vitamin E in." super 2-10 " paddy rice giant embryo gene promotor basis occurs coding strand+125bp site further and sports by G " upper No. 5, Normal University " large embryo rice that A obtains, in brown rice, total content of vitamin E improves further and reaches 52.54mg/kg (α-TE), wherein the content of alpha-tocopherol is 49.14mg/kg, be about 2.3 times of content in " super 2-10 " Brown Rice, and content of vitamin E is only 1.60mg/100g (Zhao Zesheng etc. in " Japan is fine " paddy rice, Shanghai Agricultural journal, 2002,18 (supplementary issue): 5-8).
Vitamin-E is one of most important antioxidant, has good antioxidant effect to human body, can remove the free radical in human body, delay senility.In addition, also have the display of zoology test report, vitamin-E also has obvious curative effects to hyperlipidemia prevention and therapy.Research finds, vitamin-E significantly can reduce total cholesterol, triglyceride level and low-density lipoprotein white level in diabetes mice serum, raises have provide protection to the blood fat of diabetes mouse.There are some researches prove that antioxygenation is not the bioactive unique mechanism of action of vitamin-E, vitamin-E can interact with enzyme and numerous regulatory factor thus affect the structure of cytolemma especially film fat, process (the Rimbach etc. of signal transduction in regulating cell, Proc.Nutr.Soc., 2002,61 (4): 415-425).The various small-molecule substances of recycling metabolism group methods analyst " super 2-10 " and " upper No. 5, Normal University " two kinds of Brown Rices.Find in " upper No. 5, Normal University " Brown Rice; except GABA content is apparently higher than except " super 2-10 " paddy rice, liver is had to the Oligomeric maltose content of the reduced glutathion of defencive function and applicable patients with diabetes mellitus is also obvious improves (table 3) than " super 2-10 " paddy rice.According to report displays such as Maeda, the GABA content in " Japan is fine " Brown Rice is approximately only 1mg/100g (Maeda etc., Breeding Science, 2001,51 (3): 211-213).
Table 3, " super 2-10 " and " upper No. 5, Normal University " two kinds of Brown Rice part metabolite relative contents compare
" * " represents that the statistical study p value of two kinds of paddy rice same substance detected results is at least less than 0.05 level.
Gsh is by the tripeptide compound of L-glutamic acid, halfcystine and Formation of glycine, is divided into reduced glutathion (GSH) and Sleep-promoting factor B (GSSG) two class.Gsh has extensive use clinically: as antioxidant, can Cell protection film, prevents erythrocyte hemolysis and promotes the reduction of methemoglobin; Can play a protective role to symptoms such as the oligoleukocythemia caused by radioactive rays, radiopharmaceuticals or chemotherapy; To anoxenia, feel sick and hepatic diseases caused by discomfort there is mitigation; Can be used for the antidotal therapy of carbon monoxide, heavy metal and organic solvent poisoning.Also found that GSH may have the effect suppressing hiv virus in recent years.As in body to a kind of important antioxidant of antioxidant radical, gsh is significant for clinical treatment, there is provided the antioxidant of q.s can improve the prognosis of patient, the long inflammatory reaction of obvious reduction urgent patient (Fan Yue equality, Chinese Clinical nutrition magazine, 2003,11 (2): 136-139).Reduced glutathion can also as hepatic.When human liver's damaged, by using reduced glutathion class medicine, as Tai Te, the liver just can helping damaged by supplementing reduced glutathion carries out self-regeneration, plays liver protection function.
Oligomeric maltose, there is easy to digest, low sugariness, hyposmosis characteristic, energization time can be extended, there is functions such as strengthening human body endurance, antifatigue, easily there is a series of physiological change and the symptoms such as dehydration, fever, myoneural dromotropism, functional disorders of brain in human body, and after edible Oligomeric maltose, can not only keep glucose level after overweight (or large) physical demands and long strenuous exercise, reduce the generation of blood lactase acid, and insulin secretion balance.Human trial proves, after using oligose, endurance and function power can increase by more than 30%, and effect clearly.Oligomeric maltose can be used on produce be applicable to diabetics without in sucrose food, add various beverage, milk-product, nicy, drink to as suitably replaced part sucrose; Add in the varieties of food items such as various jam, can, the various drinks of sausage, produce all kinds of sugar-free, be applicable to the specific protective foods of diabetes patient.HUMAN HEALTH is all things basis, and health more can promote " Happiness Index "." doctor food homology ", the saying of " integration of drinking and medicinal herbs " is had in traditional Chinese medicine.Paddy rice is the most important food crop of China, and rice is at least the staple food grain on China 1/2 population dining table.Giant embryo gene of the present invention can make rice be rich in the nutritive substance such as gsh, Oligomeric maltose, strengthens the healthy effect of eater by contributing to reaching.
Embodiment 2
Embodiment 2 utilizes giant embryo gene ge of the present invention by mainly describing
ccultivate the various method that there is giant embryo phenotype, contain multiple important nutrient new rice variety simultaneously in brown rice.Giant embryo gene type GG in common embryo paddy rice is represented, containing ge
cgiant embryo gene type gg in Characters of Giant Embryo Rice " upper No. 5, Normal University " paddy rice represents.First common embryo paddy rice (GG) hybridized to obtain F1 seed and plant with large embryo rice in embodiment 1 " upper No. 5, Normal University " (gg) and obtain F1 plant, F1 plant giant embryo gene type is Gg.The selfing of F1 plant obtains F2 seed, and plantation F2 seed also obtains F2 plant.According to giant embryo gene hereditary property, the giant embryo gene type of F2 plant has three kinds: GG, Gg and gg.
Get F2 plant leaf, extract STb gene, carry out giant embryo gene amplification, then check order.The order-checking of different F2 plant giant embryo gene has three kinds of results.(1) on a pair that have the 7th homologous chromosomess, two giant embryo allele encodes chain initiating codons, first deoxynucleotide upstream-777 is identical with " Japan is fine " paddy rice with between-778, adenyl-deoxyribonucleotide is not all had to insert, be simultaneously all guanine deoxyribonucleoside acid (G) at giant embryo gene coding strand initiator codon first deoxynucleotide downstream+125 place, the giant embryo gene type of correspondence is GG type like this, (2) on a pair that have the 7th homologous chromosomess, a giant embryo allele encodes chain initiating codon first deoxynucleotide upstream-777 is identical with " Japan is fine " paddy rice with between-778, adenyl-deoxyribonucleotide is not had to insert, also be simultaneously guanine deoxyribonucleoside acid (G) at giant embryo gene coding strand initiator codon first deoxynucleotide downstream+125 place, but it is identical with " upper No. 5, Normal University " paddy rice with between-778 in another giant embryo allelic coding strand initiator codon first deoxynucleotide upstream-777, adenyl-deoxyribonucleotide is had to insert, be simultaneously adenyl-deoxyribonucleotide (A) at giant embryo gene coding strand initiator codon first deoxynucleotide downstream+125 place, the giant embryo gene type of such correspondence is Gg type, (3) on a pair that have the 7th homologous chromosomess, two giant embryo allele encodes chain initiating codons, first deoxynucleotide upstream-777 is identical with " upper No. 5, Normal University " paddy rice with between-778, all there is adenyl-deoxyribonucleotide insert, be simultaneously all adenyl-deoxyribonucleotide (A) at giant embryo gene coding strand initiator codon first deoxynucleotide downstream+125 place, the giant embryo gene type of correspondence is gg type like this.Observe three kinds of seed bearing brown rice of genotype, (1), (2), (3) three types plant are respectively the separative brown rice of normal chick embryo brown rice, normal chick embryo and giant embryo isozygotied and the giant embryo brown rice isozygotied.
The seed that Retention Type (1) and (3) plant are born, then the selfing plantation carrying out 4-5 generation can obtain the normal chick embryo paddy rice and large embryo rice new variety of isozygotying.
The content of vitamin E of large embryo rice brown rice and normal chick embryo Brown Rice is detected by high performance liquid phase instrument, the relative content passing through the GABA of metabolism group method detection large embryo rice brown rice and normal chick embryo new rice variety brown rice, gsh and various Oligomeric maltose again finds, the content of the various nutritive substances in large embryo rice brown rice is all significantly more than normal chick embryo paddy rice.
In natural colored Brown Rice, as red rice epidermis, containing the important member in flavonoid, cyanidin(e) is combined the anthocyanogen formed with glycosyl.Flavonoid has various health care functions as a class natural bioactivity substance to human body, and it is strengthening body immunity and the effect prevented and treated in multiple chronic disease etc. is more and more subject to people's attention.As far back as 2000, Middleton etc. reported (Middleton etc., Pharmacol Rev., 2000,52 (4): 673-751), and Flavonoid substances has good effect to resistance mammalian cell inflammation, heart trouble and cancer aspect.Flavonoid substances also have anti-oxidant, reduce serum cholesterol and serum lipid, anticancer growth and physiological function (Lazze etc., Mutat.Res., 2003,535 (1): 103-115 such as anticancer; Russo etc., J.Nutr.Biochem., 2005,16 (1): 30-37).The picked-up of flavonoid food can reduce the death risk (Mink etc., Am J.Clin.Nutr., 2007,85 (3): 895-909) being caused people by coronary heart disease, cardiovascular disorder and a variety of causes.The research of McCullough etc. (McCullough etc., Am J.Clin.Nutr., 2012,95 (2): 454-464) shows, even if the food taking in the enrichment of minute quantity flavonoid is also of great advantage to human body.
In addition, also containing VITMAIN B1, B2, fat, the food fibre (Wu Guoquan etc. useful to people in red rice, China's rice, 2005,, and have extremely abundant amino acid and mineral element (Wu Shizhao etc., Agricultural University Of South China's journal 7 (5): 15-15), 1996,17 (3): 19-24).Trace elements iron in red rice, zinc, germanium, selenium are all higher than common rice.Red rice except containing except abundant nutritive substance, disease that the resistance of oxidation and preventing and treating that can also improve body is caused by oxyradical (horse is waited quietly, Food science, 1999, (10): 54-55; Chen Qixuan etc., Journal of Nutrition, 2001,23 (3): 246-248).
Red rice cream-coloured by two pairs of genes, Rc and Rd determines.These two pairs of genes all carry out heredity by single-gene, but have interaction again simultaneously.When Rc and Rd exists and isozygoty simultaneously, namely genotype is RcRcRdRd, cream-colouredly stably could show as redness (Eiguchi etc., Rice Genetics Newsletter, 1995,12:87-189; Furukawa etc., Plant J., 2007,49 (1): 91-102).
In the present embodiment, can also select to have red rice parental rice with containing ge
ccharacters of Giant Embryo Rice " upper No. 5, Normal University " paddy rice hybridizes, and utilizes above-mentioned same method to obtain the plant of (1), (2), (3) three kinds of giant embryo gene types.The DNA of (3) type plant is carried out the detection of Rc and Rd gene again, therefrom retain Rc and Rd gene and all to isozygoty the plant of (RcRcRdRd), then the selfing plantation carrying out 4-5 generation can obtain the red rice large embryo rice isozygotied.
In the present embodiment, by there is characteristic and the giant embryo of red seed coat and combining containing multiple important nutrient characteristic, cultivate containing ge
cthe giant embryo red rice paddy rice of new giant embryo gene, can obtain the new rice variety that nutrition and health care function is higher.
Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention, as select with containing ge
cthe parent that Characters of Giant Embryo Rice " upper No. 5, Normal University " paddy rice carries out hybridizing can also be fragrant rice or black or purple or the various paddy rice kind such as brown.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.
Claims (10)
1. a gene order, it is characterized in that, described gene order comprises following feature: insert an adenyl-deoxyribonucleotide between upstream-777 and-778 deoxynucleotide of first deoxynucleotide of Os07g0603700 genes encoding chain first codon; Downstream+125 place of first deoxynucleotide of Os07g0603700 genes encoding chain first codon is adenyl-deoxyribonucleotide by guanine deoxyribonucleoside acid mutation.
2. gene order according to claim 1, is characterized in that, described gene order at least comprises nucleotide sequence shown in SEQID No.1 or the nucleotide sequence with SEQ ID No.1 sequence at least 90% homology.
3. gene order according to claim 2, is characterized in that, described gene order is nucleotide sequence shown in SEQ ID No.1 or the nucleotide sequence with SEQ ID No.1 sequence at least 90% homology.
4. an application for gene order as claimed in claim 1, is characterized in that, comprising: described gene order be implanted in Oryza (Oryza) recipient plant cell genome.
5. application according to claim 4, it is characterized in that, described gene order is implanted in oryza plant recipient cell genome together with the second gene order, and described second gene order is to bring other proterties for plant but not affect other gene orders of said gene functional nucleotide sequence.
6. application according to claim 4, is characterized in that, described oryza plant is selected from: Asian Cultivated Rice (Oryza sativa), Oryza glaberrima Steud (Oryza glaberrima), short tongue wild-rice (Oryza barthii), particle wild-rice (Oryza granulata), open up clever wild-rice (Oryza glumaepatula), wide leaf wild-rice (Oryza latifolia), long stamen wild-rice (Oryza longistaminata), south wild-rice (Oryzameridionalis), Buddhist nun's vara wild-rice (Oryza nivara), oryza officinalis (Oryza officinalis), Oryza punctata (Oryza punctata), common wild-rice (Oryza rufipogon), the general wild-rice of horse basket (Oryza malapuzhaensis), Oryza minuta (Oryza minuta), root stock wild-rice (Oryzarhizomatis), Oryza eichingeri (Oryza eichingeri), high―strong nurserystock (Oryza alta), large sterile lemma wild-rice (Oryza grandiglumis), Australia wild-rice (Oryza australiensis), long sterile lemma wild-rice (Oryza longiglumis), Malaysia wild-rice (Oryza ridleyi), oryza meyeriana (Oryzameyeriana), short flower pesticide wild-rice (Oryza brachyantha), extremely short grain wild-rice (Oryzaschlechteri), Oryza coarctata, and the improving the breed arbitrarily of above-mentioned kind.
7. application according to claim 6, is characterized in that, described in improve the breed be selected from hybridization, backcross in any one or a few.
8. according to the application in claim 4-7 described in any one, it is characterized in that, described oryza plant is Color rice.
9. improve a method for Oryza (Oryza) plant trait, it is characterized in that, comprising: gene order described in claim 1 is implanted in described oryza plant recipient cell genome.
10. method according to claim 9, is characterized in that, described implantation is realized by any one or a few in hybridization, baclccrossing techniques.
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| CN114231540A (en) * | 2022-01-05 | 2022-03-25 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Application of Chinese wild rice ZLRc gene in improving content of procyanidine in rice seeds |
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| CN113403336A (en) * | 2021-08-03 | 2021-09-17 | 广东省农业科学院水稻研究所 | Method for cultivating giant embryo japonica rice variety by editing rice giant embryo gene GE |
| CN113403336B (en) * | 2021-08-03 | 2021-11-19 | 广东省农业科学院水稻研究所 | Method for cultivating giant embryo japonica rice variety by editing rice giant embryo gene GE |
| CN114231540A (en) * | 2022-01-05 | 2022-03-25 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Application of Chinese wild rice ZLRc gene in improving content of procyanidine in rice seeds |
| CN115094067A (en) * | 2022-05-07 | 2022-09-23 | 中国水稻研究所 | Rice nutrient quality regulation gene OsVE1 and application of encoded protein thereof |
| CN115094067B (en) * | 2022-05-07 | 2024-03-29 | 中国水稻研究所 | Rice nutrition quality regulation gene OsVE1 and application of protein encoded by same |
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