CN1128881C - Method for improving quality of gramineous crops - Google Patents

Abstract

The present invention discloses a method for improving the quality of gramineous crops, which comprises the steps that cDNA cloned from potatoes for encoding high-lysine protein and a 19Z promoter specifically expressed by maize are constructed to form a plant expression carrier for transforming gramineous plants, such as maize, so as to obtain gramineous plant strains with the obviously enhanced content of protein and lysine in seeds.

Description

The method of improvement Gramineae thing crop quality

The present invention relates to improve the method for quality of gramineous crops, specifically utilize the kind of plant gene engineering technology improvement crop, promptly utilize the high-lysine protein gene transforming gramineous crop, thereby improve protein and lysine content in the seeds of gramineous crops.

Along with the increase of agricultural products in China output and the raising of living standards of the people, the problem of staple crops inferior quality is more outstanding, causes a large amount of of hybrid rice and cotton to overstock, and does not only satisfy the needs of living standards of the people, world market more difficult to get access.And utilize plant genetic engineering improvement crop alimentary and processing quality to become one of research focus in the world, its application potential not only is to improve the value of primary agricultural products itself, more relates to a plurality of links that the reduction of tooling cost, the simplification of complete processing, the raising of product market competitiveness etc. are associated.Methionin is as people and the necessary amino acid of monogastric animal, and content is very low in the seed of main cereal crop such as corn, paddy rice, wheat, becomes main limiting amino acid, has had a strong impact on its nutritional quality.

By the lysine content of genetically modified approach raising crop seed, mainly contain the progress of following two aspects at present:

1. artificial reconstructed or design high-lysine protein gene: pioneer species Subsidiary Company (Pionner Hi-bredInc.) is once by transforming natural genes encoding, with the part non-essential amino acid in the alternative native protein of indispensable amino acid, thereby reach the purpose that improves essential amino acids content, utilize the designed high-lysine protein gene of this method, about 14% (weight ratio) of its encoded protein lysine content.Pioneer species Subsidiary Company has patent (WO9410315) to this research method.In addition, the work of the de novo synthesis that the minority document is reported (de novo synnthesis) high-lysine protein gene, be to design the non-existent gene of nature fully, be difficult to the proteic characteristic that fullys understand that it is coded, only on the seed of transgenosis model plant (tobacco), obtain to express (Othani et al., 1991 at present; Keeler et al., 1997).Carry out the international monopoly retrieval and do not find the patent of relevant de novo synthesis high-lysine protein gene.

2. natural high-lysine protein gene

(Haiwaii Uni.) has patent (WO9707665) to a high-lysine protein gene of being cloned from Semen Psophocarpi tetragonolobi in the University of Hawaii.The albumen lysine content of this coded by said gene is about 11% (weight ratio).

The cDNA fragment that the inventor clones from potato pollen, its encoded protein matter lysine content reaches 19% (weight ratio), is higher than reported so far natural far away or through transformed lysine-rich protein.This cDNA and corn 19Z seed specific expression promotor are made up the plant expression vector maize transformation, lysine content is up to 0.48% of seed dry weight in transgenic corn seed, protein reaches 15% of seed dry weight, and high-lysine protein gene can genetic stability, R2, according to improving 25-35%, improve more than 50% by indivedual plant for Methionin average content comparison in the strain system for R3, protein improves 10%, and indivedual plant improve 38%.

The objective of the invention is to utilize the quality of transgenic technology improvement gramineous crop, promptly by will being transformed into grasses such as corn, paddy rice, to obtain the Gramineae plant that protein and lysine content obviously improve in the seed from the cDNA of the coding lysine-rich protein of potato pollen.

The accompanying drawing summary

Fig. 1 .PCR product electrophoresis detection: laneA; The PCR product; LaneB; The DNA/HindIII/EcoRl molecular weight standard;

Fig. 2. the structure of plant expression vector p19zKH;

Fig. 3. the screening of kanamycin-resistant callus tissue and differentiation, corn kanamycin-resistant callus tissue after six weeks of Fig. 3 A. screening; The differentiation of Fig. 3 B. kanamycin-resistant callus tissue is sprouted;

Fig. 4. corn regeneration plant, corn regeneration plant in Fig. 4 A. greenhouse; Fig. 4 B. regeneration plant knot fringe;

Fig. 5. the Southern hybridization of transgenic corns, Fig. 5 A.R0 is for the Southern hybridization of regeneration plant; Fig. 5 B.R1 is for the Southern hybridization of regeneration plant; Fig. 5 C.R2 is for the Southern hybridization of regeneration plant;

Fig. 6 .R2 is for the Northern hybridization of transgenic corn seed;

Fig. 7 .R2 is for the Western hybridization of transgenic corn seed;

Table: the genetic stability analysis of lysine content in the transgenic corn plant;

The present invention realizes according to method as described below:

With the cDNA SB401 and the Pollen Maydis specifically expressing 19Z promotor of the coding lysine-rich protein of specifically expressing in the potato pollen, constitute plant conversion carrier from 3 ' the Transcription Termination zone of rouge alkali synthetase (no) gene.This carrier is bombarded maize calli with particle gun, the milpa that protein and lysine content improve in the acquisition seed.

Utilize that lysine content is up to 0.48% of seed dry weight in the transgenic corn seed of method of the present invention preparation, protein reaches 15% of seed dry weight, and high-lysine protein gene can genetic stability, R2, R3 for Methionin average content comparison in the strain system according to improving 25-35%.

Embodiment 1 utilizes polymerase chain reaction (PCR) amplification Pollen Maydis specifically expressing 19Z promotor;

Pollen Maydis specifically expressing 19Z promotor obtains from corn gene group DNA with polymerase chain reaction (PCR).The nucleotide sequence of corn 19Z promotor is delivered (Matzke et al. (1993) Plant Mol.Biol.22:553-554), the Oligonucleolide primers that can be designed for polymerase chain reaction (PCR) by this sequence this promotor that increases.Oligonucleolide primers is:

SEQ?ID?Nol：5’TCCTAAGCTTCTTCCTAGTGTT3’；

SEQ ID No2:5 ' TGTTGGTACACTATTGTGCTT3 '; Amplified reaction carries out in the 50ul system, contains the 50ng template, 5ul 10XPCR damping fluid, 4ul dNTP (2.5mM), 5 ' end and 3, end bow each 2ul of thing (10uM), 0.ul Tag enzyme (5U/ul) and ultrapure H2O.Amplification condition is 94 ℃, 1min; 55 ℃, 1min; 72 ℃, 2min; Totally 30 circulations are extended 10min at 72 ℃ at last.Reaction product is through 0.8% agarose gel electrophoresis, and uses ethidium bromide staining, shows that the intensive DNA of about 700bp is with, and this size is the expection size (Fig. 1) of corn 19Z promotor.

The fragment that this PCR produces is carried out terminal polishing with E.coli T4 archaeal dna polymerase, and be inserted into pUC19 SmaI site, obtain positive colony pCS1, extract plasmid and on ABI-370 nucleic acid automatic sequencer, carry out sequential analysis, full sequence is total to 694bp as a result, this sequence is shown in SEQ ID No.3 (Liang Hua etc., (1996) biotechnology journal 12 (3): 295-300).SEQ?ID?No3：

TCCTAAGCTTCTTCCTAGTGTTTTTTGTTGTGATTGAGTCGACACAGCAACAACACTGCACTAT

TACAACCAGTACGACTATATCAACTAGCAATGTCTTCCTTATATGTTACTATTTATTTTGCTCA

TATTCATTATGTTTAAATCACATAGGCACCTTTCTATTGGCTTCAAAAAATTAGTATCAACTTT

CTAGATTAAAATGAAACTAAAAGTACATAAATTTCTATCGGTGGGGAACGAGTGATTCTTTAAA

CCGATTATTACACAAGTTAACCACACTAAAATTAACATTGGTGAATCGTGCCATGATTTTTTTT

TAGTGGAAAATAGCCAAACCAAGCAACACATATGTGGCTATCCTTACACATGTGTAAAGGTATT

GCATCACACCATTGTCACCCATGTATTTGGACAATACCGAGAGGAAAAACCACTTATTTATTGT

ATTTTATCAGTTTATCTTGCTTACGTATAAATTATAACCCAACAAAGTAATCACTAAATGTCAA

AACCAACTAGATACCATGACATCTCTACCTTATCTTACTAATATTCTTTTTGCAAAATCCAAAA

GTAATCTTGCACAAGCACAAGGACTGATATGTGTATAAATATCTCTTAGATTAGTAGTTAATAC

ATCACTCATATTAAGACCAACTAGCAACATAGAAAGCACAATAGTGTACCAACA

The clone of embodiment 2 potato high-lysine protein genes

Potato (Solanum berthaultll) pollen construction cDNA library with external sprouting; Utilize the pollen of sprouting; Mature pollen; The cDNA probe of blade and flower tissue (not containing flower pesticide) screens the library; has obtained the cDNA ( called after SB401 ) of specifically expressing in blade and pollen.Its nucleotide sequence total length is 1036bp, and initiator codon is shown in SEQ ID No.4 ( Liu etal. ( 1997 ) Plant Mol.Biol.33:291-300 ) to the sequence between the terminator codon.SEQIDNo 4：1 ATGGGTTGTG GGGAATCAAA GCACGCAGTT GCAACGGAGA ACGCCACGAT TCCTAAGAAC61 AAGAGATCAT TGAGTTCTAA ATCCGAATCC ACAAAGGGTG AAAATGTCGT AAAAACTGAA121 AATGGGGTTG GTAGTGATGA AAAAGTGGAG GAGGAGAAGG AGTTGATTGC ACCGAAAGTG181 GTGGCTGTGG AAAAAGAGAA GTCTGAGAAG AAAGAGATGG TGGAATTGGA AAAGGCGAAA241 GAAGATGAGG TTGTTGAAAA GAAAGAAGAG AAAGTTGTAG AGACGAAGAA TGAAACAATC301 CATGTTGCTG TTGTAGAGAA GAAGAATGAA AATGATGAAA CAACAACCCC TGTTTCTGTT361 ATAGAGAACG ATGAAACAAC TCCGGTTGCT GTTGTAGAGA AGAAGAATGA AAATGAGGAA421 ACAGTCCCTG TTTCTGTTGT TGCTGTTGTG GAGAAGAAAG AATCTGTTGA AGAAATTAAA481 GTAGAAGAGA AAACTGAGGA GACCATCAAG CCAATTGAAG AAGTGAAAGA CAAAGAGAAG541 GAAGAAGTTA TCGCTATTTC TGAGGCCACA GATGCTACTA AACCAGAAAC TGTCAAGGAT601 GATGATAAAC CAGAGACAGA GGAAAAGCCA AAAGAGGAGG AGCAACTGAA ACAACAGCAA661 CGACAGACTC AAAAACAGAT TAAAGTGCAA GCATGGGAAT ATGGAAGGAA GAGTGTTTAT721 TAG3SB401：

Expression cassette is made up of from 3 ' the Transcription Termination zone (Depickere et al. (1982) J.Mol.Appl.Genet.1:561-570) of rouge alkali synthetase (no) gene Pollen Maydis specifically expressing 19Z promotor (SEQ ID No.3).The plasmid called after p19zK that will contain this expression cassette.Whole expression cassette upstream is HindIII, and Xbal site, downstream are the EcoRI sites.Selectable marker gene is a hygromycin phosphotransferase gene, its aminoglycoside 4-phosphotransferase APH (4)-I that encodes.

Concrete building process is shown in Fig. 2: the 35S promoter with 19Z promotor replacement Rok219, again high-lysine protein gene SB401 is inserted between 35S and the Nos terminator, and obtain plasmid p19zk.To contain hygromycin selectable marker gene plasmid pHyg (Gritz, L and Davies are J.1983) and cut with the HindIII enzyme, and behind the Klenow polishing, reclaim the 2.0kb fragment, the EcoRI enzyme that is connected to p19zk is cut, and the site behind the Klenow polishing makes up and obtains p19zKH.

The Rok219 preparation process: plasmid pBI121 (available from Clontech company) uses the HindIII/XbaI double digestion, with the fragment that obtains is that 35S promoter is inserted into plasmid pUC19 (Messing J.1983) HindIII/XbaI site, obtain plasmid pUC35S, with pBI121 SacI/EcoRI double digestion, the fragment that obtains is inserted into the SacI/EcoRI site of plasmid pUC35S, obtains plasmid Rok219.

Embodiment 4 usefulness 19Z promotors/SB401 mosaic gene maize transformation

The genetic transformation of corn: get 10-12 days the female fringe in corn pollination back of sterilization, strip rataria, be put in (N6 substratum on the culture medium A, contain 2mg/L 2,4-D, 0.69g/L L-proline(Pro), 100mg/L casein hydrolysate, 2% sucrose), 27-28 ℃, dark culturing makes it dedifferentiation, form callus, with p19zKH particle gun transformed calli.BioRad Biolistic PDS-1000Helium system is adopted in the particle gun bombardment.4h before the particle gun bombardment, callus is cut into small pieces, transfer to the culture medium A that contains 0.4M N.F,USP MANNITOL, after the bombardment, callus is gone up at high glucose medium (culture medium A that contains 0.4M N.F,USP MANNITOL) continue overnight incubation, transfer to then and cultivate after 4-6 days on the culture medium A, be transferred to contain on Totomycin (20mg/L) the resistance culture medium A and cultivate, weekly subculture once, after 2 months, be transferred to division culture medium (the N6 substratum contains 5% sucrose) and go up regeneration induction (Fig. 3,4).The Molecular Detection of regeneration plant

The PCR of aftergrowth and Southern analyze: the trace of rotaring gene plant blade DNA extracts and the prior art technique known is adopted in the PCR operation.The PCR detection uses the primer according to 5 ' and 3 ' the terminal sequence design of high-lysine protein gene (SB401) to carry out.The DNA that extracts transgenic corns and contrast blade increases, total DNA of PCR male regeneration plant cut with the HindIII enzyme, with ³²The SB401 of P-mark is as probe, and crossover operation adopts the prior art technique known.The presentation of results foreign gene has been integrated into (Fig. 5) in the corn gene group.

The extraction of RNA and proteinic detection: RNA and Northern analysis operation adopt the prior art technique known in the transfer-gen plant seed, with ³²The SB401 effect probe of P-mark is hybridized, and the result shows that high-lysine protein gene accumulates (Fig. 6) in transgenic corn seed.

Utilize the antiserum(antisera) of lysine-rich protein to carry out Western hybridization, the result shows lysine-rich protein stable accumulation (Fig. 7) in corn seed.The analysis of Methionin and protein measuring and genetic stability in the transgenic corns.

Methionin and protein content determination are carried out in the solid back of transfer-gen plant (Ri is for seed), select the high plant seed of content by plant plantation (R2 generation), obtain R3 generation according to this, and R2 and R3 are measured (table 1) for Methionin in the seed and protein content.Result's lysine content in transgenic corn seed is up to 0.48% of seed dry weight, and protein reaches 15% of seed dry weight, and high-lysine protein gene can genetic stability.R1 is high for content, and lysine content is also high in its offspring's strain system.And R2, according to improving 25-35%, improve more than 50% by indivedual plant for Methionin average content comparison in the strain system for R3, and protein improves 10%, and indivedual plant improve 38%.Methionin and protein content determination entrust grain quality supervision and inspection center of the Ministry of Agriculture to carry out.

Reference:

Liu?JQ，Seul?U?and?Thompson?R(1997)Cloning?andcharacterization?of?a?pollen-specific?cDNA?encoding?a?glutamic-acid-rich?protein(GARP)from?potato?solanum?berthaultii.Plant?Mol.Biol.33：291-300.

Matzke?AJM，Stoger?EM，Matzke?MA?et?al.(1993)Plant?Mol.Biol.22：553-554.

Othani?et?al.(1991)Normal?and?lysine-containing?zeins?areunstable?in?transgenic?tobacco?seeds.Plant?Mol.Biol.34：15-29.

Liang Hua, Ma Chonglie, Zhao Qian, Ao Guangming (1996) corn zein gene promotor RCR amplification and the expression of driving gus gene in the transgene tobacco seed thereof.Biotechnology journal 12 (3): 295-300.

Gritz，L，Davies，J.(1983) Plasmid-encoded?hygromycin?Bresistance?the?sequence?of?hygromycin?B?phosphotransferase?geneand?its?expression?in?Escherichia?coli?and?Saccharomycescerevisiae.Gene?25：179-188.

Messing?J.(1983)New?M13?vectors?for?cloning.Methods?Enzymol.101：20.

Depickere?et?al.(1982)J.Mol.Apll.Genet.1：561-570.

Table

(1) lysine content in the transgenic corn plant

R ₁For lysine content in the plant

Lysine content g/100g dry seeds	0.35～0.37g	0.38	0.42	0.48	＜0.35
						Increase rate	10～20％	20～30％	30～40％	More than 40%	Do not reach 10%
The strain number	17	7	1	1	44
						Percentage	24％	10％	1.4％	1.4％	63.2％

Detect altogether transfer-gen plant 70 strains, lysine content improves 26 strains more than 10%, accounts for 36.8%.

                R ₁Content for total protein in the plant

Protein content g/100g dry seeds	11.1～12.9	12.9～13.85	13.85～15.0	＜11％
					Increase rate	10～20％	20～30％	More than 30%	Do not reach 10%
The strain number	19	16	8	27
					Percentage	27.1％	22.9％	11.4％	38.6％

Detect altogether several 70 strains of transfer-gen plant, total protein improves 43 strains more than 10%, accounts for 61.4%.

                 R ₁Relation for lysine in the plant and protein

Lysine and albumen improve situation	Lysine and albumen improve more than 10% simultaneously	Lysine improves more than 10%, and protein does not reach 10%	Protein improves more than 10%, and lysine does not reach 10%	Lysine and protein all do not reach 10%
					The strain number	20	6	25	19
Percentage	28.6	8.6	35.7	27

Lysine and protein is totally 51 strains that improve more than 10% wherein, account for 72.9%. (2) genetic stability

        R ₂、R ₃For lysine content in the plant

Strain		R 2、R 3For lysine content
				Content (g/100g dry seeds)	Bring up to %
		1 Q31 * Z31 19Z promoter	90..4(R 1)   CK			0.48         0.33	154.8     100
90-4-1(R 3) the average the highest CK of individual plant	0.39         0.46         0.29			134.5     158.6     100
			2 Z31 * Q31 19Z promoter		34-5(R 1)   CK	0.42         0.33	135     100
34-5(R 2) the average the highest CK of individual plant	0.37         0.38         0.29	127     131     100
				3 Z31 * Q31 35S promoter	64-2(R 1)   CK	0.38         0.33	122.6     100
64-2(R 2) the average the highest CK of individual plant	0.41         0.42         0.29	138     144.8     100
			4 Z31 * Q31 19Z promoter		BZ-23-3(R 1)   CK	0.37         0.31	119.4     100
BZ-23-3(R 2) the average the highest CK of individual plant	0.41         0.44         0.29	141.4     157.7     100
				5 Z31 * Q31 19Z promoter	BZ-23-4(R 1)   CK	0.37         0.29	127     100
BZ-23-4(R 2) the average the highest CK of individual plant	0.37         0.44         0.29	127     157.7     100

                       R ₂、R ₃For protein content in the plant

Strain		R 2、R 3For protein content
				Content (g/100g dry seeds)	Bring up to %
		1 Q31 * Z31 19Z promoter	90-4(R 1)     CK			15.04       10.43	139     100
90-4-1（R 3) the average the highest CK of individual plant	11.94       13.351       11.295			105.7     118.2     100
			2 Z31 * Q31 19Z promoter		34-5(R 1)     CK	11.56       10.43	110.3     100
34-5(R 2) the average the highest CK of individual plant	12.48       12.852       11.295	110.5     138     100
				3 Z31 * Q31 35S promoter	64-2(R 1)     CK	12.08       10.43	115     100
64-2(R 2) the average the highest CK of individual plant	12.31       13.33       11.295	110     118     100
			4 Z31 * Q31 19Z promoter		BZ-23-3(R 1)     CK	14.28       10.90	131.0     100
BZ-23-3(R 2) the average the highest CK of individual plant	13.02       13.73       10.74	121.22     127.8     100
				5 Z31 * Q31 19Z promoter	BZ-23-4(R 1)     CK	12.14       10.92	112.0     100
BZ-23-4(R 2) the average the highest CK of individual plant	11.31       12.71       10.74	105.3     118.3     100

Claims (4)

1. improve the method for quality of gramineous crops, comprising:

A. the cDNA of the lysine-rich protein that will clone from potato and promotor make up plant expression vector;

B obtains Methionin and the obvious plant of improving of protein content in the seed with the transforming gramineous plant of expression vector that obtains.

2. method according to claim 1, wherein said promotor are the 19Z promotors of specifically expressing in the corn seed.

3. method according to claim 1, wherein said grass comprises corn, paddy rice.

4. method according to claim 1, wherein said lysine-rich protein cDNA is SlB401.

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