CN1552874A - Method for producing rice seed with protein concentration improvement - Google Patents
Method for producing rice seed with protein concentration improvement Download PDFInfo
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- CN1552874A CN1552874A CNA031290035A CN03129003A CN1552874A CN 1552874 A CN1552874 A CN 1552874A CN A031290035 A CNA031290035 A CN A031290035A CN 03129003 A CN03129003 A CN 03129003A CN 1552874 A CN1552874 A CN 1552874A
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Abstract
This invention relates to production of rice seeds containing high protein content. It is carried out by: 1) transforming rice with expression carrier containing wax gene reverse sequence, 2) growing the said rice to determine its progeny, and 3) determining the descendant seeds containing high protein content. It is a novel way.
Description
Technical field
The invention belongs to the crop breeding field, specifically, the present invention relates to the production method of the rice paddy seed of protein content raising.
Background technology
In rice quality, the food flavor quality is a very important aspect.People such as Capamang (Capampang, C.B. etc., Sci.Fd.Agri, 1973,24:1589-1594; Juliano, B.O., Cereal Sci.Today, 1971,16:334-338,340,360; Khush, G.S. etc., Chemical Aspects of Rice Grain Quality, 1979:21-32) several indexs of rice food flavor quality are estimated in proposition once: amylose content, gelatinization point and glue denseness etc.In the many index of estimating rice food flavor quality, the height of amylose content is a most important index.Rice grain amylose content is high more, and the rice bloated property of cooking is big, and meal is dry and soft and look light, and cold back matter is hard, and food flavor is poor more; The rice bloated property of cooking of low amylose content is little, and meal is wet sticking, soft and glossy, and food flavor is better relatively.
Protein is the basis of life, and it has important role to human body.Because development imbalances, people's diet formula of different areas is widely different.Replenish protein and be the poverty-stricken area of staple food with rice from animal food being difficult to, the protein source in people's food just can only be taken from rice; In Economic development big city faster, though there has not been the insufficient problem of protein uptake, the increasing diabetic subjects in big city need high protein, low carbohydrate staple food; For some can not edible animal proteins the special disease patient, also need market that the fine vegetable-protein can be provided.
The leading indicator of rice nutrition quality is a grain protein content.Though a tree name reports that common grain protein content is lower, generally only be about 8% (Juliano etc., Cereal Science Today, 1968:299-313; Juliano etc., Journalofthe Science of Food and Agri-culture, 1973,24 (1): 295-306; Boulter etc., Nucleic Acidsand Proteins in Plants I, Berlin, Springer-Verlag, 1982; Zhen Hai etc., Agricultural University Of South China's journal, 1997,18 (14): 16-20), but compare with main cereal crop such as wheat, barley, corn, amino acid ratio of components in the protein in rice corn is than balance, and protein value is higher, is easy to be absorbed by human consumption, belong to fine protein (Song Wenchang etc., extraordinary rice growing and utilization, Science Press, 1997:19-20; Min Shao wipes etc., and rice breeding is learned, Chinese agriculture press, 1996,322-354; Xiong Zhenmin etc., paddy rice digest, 1992,12 (3): 1-6).Therefore, consider, be necessary to carry out the breeding of high protein paddy rice from producing quality plant protein angle.
The breeding of paddy rice high protein is subjected to very big attention at home and in the world always.In the 70-80 age in 20th century, Japanese scholar once adopted radioinduction and chemical reagent processing to attempt to improve grain protein content.There is the nineties in 20th century people to adopt the technology such as total dna direct importing of high protein species to cultivate the high protein paddy rice.Perfect along with the paddy rice transgenic technology, scholar Kyoto Univ Japan in 1999 successfully imports paddy rice with a soybean storage protein gene, in being detected, transgenic progeny shows that the grain protein content comparison is according to improving 20% (Momma K etc., BiosciBiotechnol Biochem, 1999,63 (2): 314-318).
Though these former study should still have certain effect on the raising grain protein content, but it is reported, seed protein is the proterties by the polygene coding, as only adopting a mode that imports term single gene, may be difficult to realize significantly, the purpose of general equilibrium ground raising grain protein content.
Therefore, still need the production method of the paddy rice of new protein content raising in this area.
Summary of the invention
One of purpose of the present invention provides a kind of method of producing the rice paddy seed of protein content raising.
The invention provides a kind of method of producing the rice paddy seed of protein content raising, this method comprises:
A) with the expression vector rice transformation that contains paddy rice waxy gene antisense sequences,
B) make the paddy growth that step a) obtains and gather in the crops the offspring paddy rice;
C) identify offspring's rice paddy seed that protein content improves.
The present inventor is surprised to find by research, be transformed in the paddy rice by the expression vector that will contain paddy rice waxy gene antisense sequences, not only can reduce the content of rice amylose, really can also improve simultaneously the protein content of rice, thereby realized the purpose that the mode that imports term single gene realizes significantly by adopting, general equilibrium ground improves grain protein content, provide new thinking for improveing rice nutritional quality and food flavor quality.
Description of drawings
Fig. 1 has shown binary vector p13W4 T-DNA plot structure figure, wherein: 232Wx-pro, 232Int.1: the promotor and the introne 1 of paddy rice 232 Waxy genes; Wx frag: the antisense DNA fragment (756bp) of paddy rice 232 Waxy genes; The border, the left and right sides of LB, RB:T-DNA; Nos-ter:Nos gene terminator; 35S-pro, 35S-ter:CaMV 35S gene promoter and terminator; GUS: beta-glucuronic acid Glycosylase gene; HPT: hygromycin gene; Xh:XhoI; E:EcoRI, Sa:SacI; B:BamHI; X:XbaI; S:SalI; P:PstI; H:HindIII.
Embodiment
The invention provides a kind of method of producing the rice paddy seed of protein content raising, this method comprises:
A) with the expression vector rice transformation that contains paddy rice waxy gene antisense sequences;
B) make the paddy growth that step a) obtains and gather in the crops the offspring paddy rice;
D) identify offspring's rice paddy seed that protein content improves.
In such scheme, used paddy rice is not limited to any specific kind (being).In a preferable embodiment, used paddy rice is super 2-10 japonica rice strain.
The used paddy rice waxy gene (waxy gene) of the present invention on No. 3 karyomit(e) of paddy rice, it controlling amylose starch in the endosperm synthetic (Khush etc., 1984, Genetics, 107:141-167).This gene only efficiently expresses in pollen, endosperm and the blastular of paddy rice, so it is space-time specificity expression's a gene.About concrete scheme with expression vector (as the p13W4) rice transformation that contains paddy rice waxy gene antisense sequences, can be referring to patent application publication CN1298021A, this article is all included this paper in as a reference.
The used expression vector of the present invention is not limited to any specific expression vector.The technology that makes up expression vector of the present invention also is well known to those of ordinary skill in the art, and can find in the reference such as people such as Sambrook (1989, " molecular cloning experiment guide ").
Method for transformation is to know in this area.In a preferable example, can be according to people (1994) Plant J such as for example Hiei, the method for describing among the 6271-6282 utilizes the agrobacterium tumefaciens method to transform.
After the expression vector that will contain paddy rice waxy gene antisense sequences was transformed into paddy rice, available ordinary method made paddy growth and cultivation obtain its offspring, identifies the paddy rice individual plant that the rice paddy seed protein content improves then.Can adopt routine techniques in this area about the mensuration of protein content, as micro-Kjeldahl determination (Zhu Zhancai, the mass analysis of rice wheat, Chinese food press, 1988).
Below in conjunction with embodiment the present invention is described in further detail.Yet should be appreciated that and enumerate these embodiment, and be not to be used for limiting the present invention just for an illustration.
Embodiment
Present embodiment is that the antisense waxy gene that can reduce rice grain amylose content imports paddy rice, selects the part individual plant and carry out rice grain amylose content and protein content determination from transgenic progeny.
1. test materials: the super 2-10 japonica rice that provides by the Lu Jiaan assistant researcher of Academy of Agricultural Sciences, Shanghai City crop institute; Agrobacterium strains EHA105 and binary vector p13W4 are provided by Chinese Academy of Sciences's Shanghai plant physiology and the Wang Zongyang researcher of ecological Studies institute.This carrier is to insert the Waxy-GUS fusion gene on the multiple clone site of pCAMBIA1300.Unique BamHI restriction enzyme site is arranged on this plasmid, and it is between Waxy gene promoter district and gus gene coding region.On this site, also inserted one section waxy gene BamHI fragment (from rice variety 232) (as shown in Figure 1) that 756bp is reverse.
2. test method
The screening of the cultivation of agrobacterium tumefaciens, rice conversion and resistant calli and plant regeneration are mainly with reference to method (Liu Qiaoquan etc., plant physiology journal, 1998,24 (3): 259-271) carry out of Liu Qiaoquan.
1) paddy rice rataria callus inducing and cultivating
Take away 12～15 days the immature seed (band shell) in flower pollination back and use rinsed with sterile water after 2 minutes, use 2.5%NaClO again with 70% alcohol immersion
3Sterilized 90 minutes, and used aseptic water washing 4～5 times, use scalper picking rataria in N
6D
2Evoked callus on the substratum, 26 ℃, the dark cultivation can be used for agroinfection after 4-6 days.
2) cultivation of agrobacterium tumefaciens and rice conversion
On the YEB flat board the single bacterium colony of picking one agrobacterium tumefaciens in 5ml YEB (Km 50 μ g/ml) liquid culture based on 28 ℃ of joltings (100rpm) overnight incubation.Therefrom drew 0.5ml on the 2nd day, switching is gone into 50ml AB (Km 25 μ lg/ml) liquid culture based on 28 ℃ of jolting (190～200rpm) overnight incubation.Bacterium liquid in 4000rpm centrifugal 15 minutes was removed supernatant liquor in the 3rd day, be resuspended in AAM (the AS100 μ M) liquid nutrient medium of 3/5 volume, make final bacterium liquid OD
600Value is about 0.45, is used for the genetic transformation of paddy rice.
Be ready to N
6D
2C transforms substratum, pad one deck aseptic filter paper on flat board.The rataria callus is chosen in the Φ 6cm culture dish, and the bacterium liquid resuspended with AAM soaks, and shakes frequently therebetween.After 20 minutes callus is chosen, blotted, transfer to above-mentioned N with aseptic paper
6D
2C cultivated 3 days in 26 ℃ on the substratum altogether altogether.
When cultivating altogether, except that special processing, adding the activation inductor of the Syringylethanone (AS) of 100 μ M among the culture medium A AM altogether as Agrobacterium Vir gene.
3) screening of resistant calli and plant regeneration
After rataria and Agrobacterium are cultivated 3 days altogether, change screening culture medium S over to
1Screen (rataria removes radicle), change subculture screening culture medium S after 2 weeks over to
2, the eugonic resistant calli of 4 weeks back selection is transferred to division culture medium MSRCH and is gone up differentiation (illumination 12 hours/day); The regenerated seedling is at root media 1/2MS
0The last strong plantlets and rootage of H moves into greenhouse pot culture subsequently, until forming T1 for rice paddy seed.
Utilize PCR and Southern hybridization to identify transgenic paddy rice (Sambrook J etc., molecular cloning experiment guide second edition, Beijing: Science Press, 1992), again by T1 and T2 for rice GUS colour developing (Jefferson RA., Plant Mol Biol Rep, 1987,5:387-405) result selects T2 that part antisense-Waxy isozygotys for seed, by the single-strain seed that isozygotys (brown rice) is carried out the analysis of protein content and amylose content, filter out the individual plant that protein content improves then.
Grain protein content adopts micro-Kjeldahl determination (Zhu Zhancai, the mass analysis of rice wheat, Chinese food press, 1988) to analyze.The rice grain amylose content analysis with reference to the improvement short cut technique that The Ministry of Agriculture of the People's Republic of China, MOA issues standard " NY147-88 rice matter assay method " carry out (The Ministry of Agriculture of the People's Republic of China, MOA's standard, China Standard Press, 1988, Beijing, 1-10).
Contrasting the rice amylose average content after testing is 13.4%, and the protein average content is 9.5%.In 16 the detected super 2-10 japonica rice of antisense waxy gene offsprings, 7 individual plant rice are arranged when amylose content reduces, it is obvious that grain protein content improves degree.Wherein having an individual plant rice grain amylose content to reduce obviously, be 11.4%, and its protein content is also the highest relatively, is 13.5%, and comparison is according to improving 42.11%.
The scholar of Kyoto Univ Japan once imported paddy rice with a soybean storage protein gene in 1999, transgenic progeny detects and shows that the grain protein content comparison is according to improving 20% (Momma K etc., Biosci Biotechnol Biochem, 1999,63 (2): 314-318).In the present invention's test, pass through to import the antisense waxy gene, the amplitude that the T2 that isozygotys improves for grain protein content obviously surpasses people's such as Momma K research, thereby realized as amylose content, improving the purpose of grain protein content by reducing certain composition in the rice.
Although the invention describes concrete example, having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.
Claims (3)
1. method of producing the rice paddy seed that protein content improves is characterized in that this method comprises:
A) with the expression vector rice transformation that contains paddy rice waxy gene antisense sequences;
B) make the paddy growth that step a) obtains and gather in the crops the offspring paddy rice;
C) identify offspring's rice paddy seed that protein content improves.
2. method according to claim 1 is characterized in that, described paddy rice is the super 2-10 strain of japonica rice.
3. method according to claim 1 is characterized in that, described expression vector is by agrobacterium tumefaciens method rice transformation.
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| CNB031290035A CN100516226C (en) | 2003-06-02 | 2003-06-02 | Method for producing rice seed with protein concentration improvement |
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| CNB031290035A CN100516226C (en) | 2003-06-02 | 2003-06-02 | Method for producing rice seed with protein concentration improvement |
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| CN100516226C CN100516226C (en) | 2009-07-22 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1330235C (en) * | 2005-02-03 | 2007-08-08 | 浙江大学 | Rice cultivating method for obtaining high protein seeds |
| CN100489103C (en) * | 2005-11-04 | 2009-05-20 | 上海师范大学 | Expression vector of duplicate inverted waxy gene, preparation method, and application |
| CN101250550B (en) * | 2008-04-08 | 2010-06-02 | 上海师范大学 | Expression vector changing distribution of rice storage protein as well as preparation and use thereof |
| CN102168083A (en) * | 2010-12-29 | 2011-08-31 | 中国科学院遗传与发育生物学研究所 | Method for culturing transgenic plant with decreased wax |
| JP2021110701A (en) * | 2020-01-15 | 2021-08-02 | Tdk株式会社 | Method of measuring protein content in rice |
-
2003
- 2003-06-02 CN CNB031290035A patent/CN100516226C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1330235C (en) * | 2005-02-03 | 2007-08-08 | 浙江大学 | Rice cultivating method for obtaining high protein seeds |
| CN100489103C (en) * | 2005-11-04 | 2009-05-20 | 上海师范大学 | Expression vector of duplicate inverted waxy gene, preparation method, and application |
| CN101250550B (en) * | 2008-04-08 | 2010-06-02 | 上海师范大学 | Expression vector changing distribution of rice storage protein as well as preparation and use thereof |
| CN102168083A (en) * | 2010-12-29 | 2011-08-31 | 中国科学院遗传与发育生物学研究所 | Method for culturing transgenic plant with decreased wax |
| CN102168083B (en) * | 2010-12-29 | 2013-03-20 | 中国科学院遗传与发育生物学研究所 | Method for culturing transgenic plant with decreased wax |
| JP2021110701A (en) * | 2020-01-15 | 2021-08-02 | Tdk株式会社 | Method of measuring protein content in rice |
| JP7340314B2 (en) | 2020-01-15 | 2023-09-07 | Tdk株式会社 | How to measure the protein content in rice |
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