CN102533847A - Constructing method of zein gene RNAi (Ribonucleic Acid Interference) carrier - Google Patents

Abstract

The invention discloses a constructing method of a zein gene RNAi (Ribonucleic Acid Interference) carrier. The method comprises the following steps of: constructing a recombinant carrier which is inserted into a forward target fragment and an endospem-specific expression promoter P-zp22/6 as well as a recombinant carrier which is inserted into a reverse target fragment and a terminator poly(A) by utilizing pUC19 as a frame carrier, and respectively carrying out digestion and connection on the two recombinant carriers to obtain a recombinant carrier pUC19-p22/6-22KD1-22KD2-poly(A); and inserting the GUS gene intron into the recombinant carrier to obtain the zein gene RNAi carrier of which the target fragment is partial cDNA (complementary Deoxyribonucleic Acid) fragment of a 22-KD alcohol-soluble protein gene. Another zein gene RNAi carrier is constructed by an identical method, and the target fragment of the zein gene RNAi carrier is partial cDNA fragment of a 19-KD alcohol-soluble protein gene. By adopting the method, the carrier constructing time is shortened and a transgenic plant with high lysine content can be successfully obtained.

Description

A kind of zein spirit-soluble gene RNAi construction of carrier

Technical field

The invention belongs to the plant gene engineering technology field, particularly a kind of zein spirit-soluble gene RNAi construction of carrier of suitable monocotyledons genetic transformation.

Background technology

RNA disturbs (RNA interference; RNAi) be meant endogenous or external source double-stranded RNA (double-stranded RNA; DsRNA) the specificity degraded takes place in the mRNA of the endogenous target gene of mediation, and then the expression of suppressor gene, produces the phenomenon of function corresponding phenotype disappearance.The RNAi technology has become the important method of gene functional research and crop quality improvement at present, and wherein the structure of target gene RNAi carrier is its core technology.For improving jamming effectiveness, promote the application of RNAi technology in genetic modification of plants, the RNAi construction of carrier has been carried out more research both at home and abroad.The factor that in the plant RNA interference expression vector makes up, must consider mainly contains aspect two: (1) method is easy, quick, jamming effectiveness is high; (2) in the building process to the purpose fragment less-restrictive of skeleton carrier and importing.

At present, plant RNA i carrier construction method mainly contains three kinds, comprises the RNAi carrier conventional construction method that cut-is connected to the basis with enzyme, and the plant RNA interference expression vector construction process that with the homologous recombination is the basis is as based on Gateway ^TMIt is the plant RNA interference carrier construction process that basic desmoenzyme is cut-connected that the vector construction of technology reaches with the recombinant PCR technology.In general, traditional RNAi carrier construction method length consuming time, but technological comparative maturity is applied widely; Based on Gateway ^TMThe RNAi carrier construction method of technology is a kind of simple, fast selection, be suitable for robotization, high-throughout research work, but cost is higher; If when lacking required restriction enzyme site on the skeleton carrier, more effective based on the RNAi carrier construction method of recombinant PCR technology, but should technology newer, still need and further optimize In utilizing the research of RNAi technique improvement crop quality and genetic modification of plants, the purpose that can constructed RNAi carrier successfully transform and reach improvement is another technical problem that the RNAi technology is still needed and solved.

The protein contnt of corn kernel is about 10%, wherein the 50-60% prolamine that can not absorb for people and animals.International corn wheat improvement center (CIMMYT) begins to utilize the recessive mutation gene in the seventies in 20th century Opaque-2( O2) and modifying factor content of prolamine is reduced to 22.9% by 55.1%; Make the lysine content level reach 2 times of conventional corn; And then seed selection large quantities of high-lysine corns be high-quality protein maize and widespread use in the whole world (Tan waits quietly. the high-quality protein maize Research Advances on Breeding. corn science 2006; 14 (5): 15-19); But this long (7-10), efficient is low, foresight is poor in method transformation cycle, and the high-lysine corn endosperm that in addition selects is soft and problem such as disease resistance difference, the more and more needs of incompatibility modern breeding target.The nutritive value of high-quality protein maize is equivalent to 90% of milk proteins; Many in the whole world is that the countries and regions of staple food all can be used as and substitute nutritive food with the corn, is that particularly school-ager's malnutritive problem is significant for the crowd of staple food to improving with the corn therefore.The feed that this external application high-quality protein maize is a main body is raised pigs, and, feeds chicken and can improve egg productivity more than 15% more than 30% than conventional corn feed day weight gain.Research shows; Because the prolamine particularly expression of 22-kD and 19-kD alcohol soluble protein gene has significant negative effect to lysine content; Yet traditional breeding method is difficult to find ideal high-quality germplasm; Obtain the lysine content height and the low corn variety difficulty of expressing of prolamine is very big; Major cause is that its phenotypic evaluation is that lysine content mensuration needs to use HPLC (HPLC) to analyze, because HPLC running cost is very high, is difficult in widespread use in the offspring's selection in enormous quantities in extensive germplasm screening and the breeding of new variety process.Therefore, if can pass through the RNA perturbation technique, structure RNAi carrier and conversion obtain the lysine content height and the transgenic corns of the low expression of prolamine, will be a kind of easy, quick and valid approach.

Summary of the invention

The technical problem that the present invention will solve is to overcome prior art (to utilize O2Gene and modifying factor transformation thereof obtain low and the corn variety that lysine content is high of content of prolamine) the transformation cycle is long, efficient is low, foresight is poor, and the high lysine corn varieties that selects with prior art because O2The chain effect of gene causes its seed to be defective and deficiencies such as soft endosperm and disease resistance difference; Its objective is the construction process that two zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD and pUC19-RNAi-19KD are provided; Realize suppressing the expression of zein, improve the purpose of the contents level of Methionin.

The construction process of zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD provided by the present invention may further comprise the steps:

(1) makes up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6

1. the acquisition of endosperm specificity expression promoter P-zp22/6

Genomic dna with corn variety is a template, is that primer is right with sequence shown in SEQ ID NO:1 and the SEQ ID NO:2, carries out pcr amplification, obtains the endosperm specificity expression promoter P-zp22/6 of sequence shown in SEQ ID NO:3;

2. make up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6 sequence

Cut the endosperm specificity expression promoter that has corresponding restriction enzyme site that step (1) 1. obtains with Sac I and Xba I enzyme; Be connected with the skeleton carrier pUC19 that cuts through Sac I and Xba I enzyme equally then, obtain having the recombinant vectors pUC19-p22/6 of endosperm specificity expression promoter P-zp22/6;

(2) make up the recombinant vectors pUC19-p22/6-22KD1 that inserts forward purpose fragment 22-KD-P and endosperm specificity expression promoter P-zp22/6

1. the acquisition of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

With the total RNA of corn kernel is template; With sequence shown in SEQ ID NO:4 and the SEQ ID NO:5; Carrying out the RT-PCR amplification, is template with this RT-PCR product then, is that primer is right with sequence shown in SEQ ID NO:6 and the SEQ ID NO:7; Carry out RT-PCR again, obtain the 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P shown in SEQ ID NO:8;

2. make up the recombinant vectors pUC19-p22/6-22KD1 that inserts forward purpose fragment 22-KD-P and endosperm specificity expression promoter P-zp22/6

1. the purpose fragment 22-KD-P that has corresponding restriction enzyme site that obtains with Sma I and Xba I double digestion step (2); Be connected with the same recombinant vectors pUC19-p22/6 that 2. makes up through the step (1) of Sma I and Xba I double digestion then, obtained inserting the recombinant vectors pUC19-p22/6-22KD1 of forward goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P and endosperm specificity expression promoter P-zp22/6;

(3) make up the recombinant vectors pUC19-22KD2 that inserts reverse purpose fragment 22-KD-P

Cut the purpose fragment 22-KD-P that has corresponding restriction enzyme site that step (2) 1. obtains with Hind III and Xba I enzyme; Be connected with the pUC19 carrier of cutting through Hind III and Xba I enzyme equally then, obtain inserting the recombinant vectors pUC19-22KD2 of reverse goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P;

(4) make up the recombinant vectors pUC19-22KD2-poly (A) that contains terminator poly (A)

With expression vector p3301 DNA is template, carries out pcr amplification with the terminator primer of sequence shown in SEQ ID NO:9 and the SEQ ID NO:10, obtains the terminator poly (A) of sequence shown in SEQ ID NO:11; The terminator poly (A) that further has corresponding restriction enzyme site with Xho I and Hind III double digestion; Be connected with the same recombinant vectors pUC19-22KD2 that obtains through the step (3) of Xho I and Hind III double digestion then, obtain containing the recombinant vectors pUC19-22KD2-poly (A) of terminator poly (A);

(5) structure contains endosperm specificity expression promoter P-zp22/6, the recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) of forward and reverse purpose fragment 22-KD-P and terminator poly (A)

The recombinant vectors pUC19-p22/6-22KD1 of step (2) structure and the recombinant vectors pUC19-22KD2-poly (A) of step (4) structure are used Xba I and Hind III double digestion respectively; The enzyme that will contain reverse purpose fragment 22-KD-P and terminator poly (A) is then cut product 22KD2-poly (A) and is connected on the recombinant vectors pUC19-p22/6-22KD1 that step (2) makes up; Obtain containing endosperm specificity expression promoter P-zp22/6, the recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) of forward and reverse goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P and terminator poly (A);

(6) structure of the connection of gus gene intron and zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD

With plasmid vector pGreen-0229 Backbone DNA is template, carries out pcr amplification with the gus gene intron primer of sequence shown in SEQ ID NO:12 and the SEQ ID NO:13, obtains the gus gene intron of sequence shown in SEQ ID NO:14; The gus gene intron that further has corresponding restriction enzyme site with Xba I single endonuclease digestion; Be connected with the recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) that phosphorylation processed steps (5) makes up behind Xba I single endonuclease digestion equally then; Obtain described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD, the nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD is shown in SEQ ID NO:15.

The construction process of another zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD provided by the invention may further comprise the steps:

(1) makes up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6

1. the acquisition of endosperm specificity expression promoter P-zp22/6

Genomic dna with corn variety is a template, is that primer is right with sequence shown in SEQ ID NO:1 and the SEQ ID NO:2, carries out pcr amplification, obtains the endosperm specificity expression promoter P-zp22/6 of sequence shown in SEQ ID NO:3;

2. make up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter zp22/6 sequence

Cut the endosperm specificity expression promoter that has corresponding restriction enzyme site that step (1) 1. obtains with Sac I and Xba I enzyme; Be connected with the skeleton carrier pUC19 that cuts through Sac I and Xba I enzyme equally then, obtain having the recombinant vectors pUC19-p22/6 of endosperm specificity expression promoter P-zp22/6;

(2) make up the recombinant vectors pUC19-p22/6-19KD1 that inserts forward purpose fragment 19-KD-P and endosperm specificity expression promoter P-zp22/6

1. the acquisition of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

With the total RNA of corn kernel is template; With the sequence shown in SEQ ID NO:16 and the SEQ ID NO:17; Carrying out the RT-PCR amplification, is template with this RT-PCR product then, is that primer is right with sequence shown in SEQ ID NO:18 and the SEQ ID NO:19; Carry out RT-PCR again, obtain the 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P of sequence shown in SEQ ID NO:20;

2. make up the recombinant vectors pUC19-p22/6-19KD1 that inserts forward purpose fragment 19-KD-P and endosperm specificity expression promoter P-zp22/6

1. the purpose fragment 19-KD-P that has corresponding restriction enzyme site that obtains with Sma I and Xba I double digestion step (2); Be connected with the same recombinant vectors pUC19-p22/6 that 2. makes up through the step (1) of Sma I and Xba I double digestion then, obtained inserting the recombinant vectors pUC19-p22/6-19KD1 of forward goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P and endosperm specificity expression promoter P-zp22/6;

(3) make up the recombinant vectors pUC19-19KD2 that inserts reverse purpose fragment 19-KD-P

Cut the purpose fragment 19-KD-P that has corresponding restriction enzyme site that step (2) 1. obtains with Hind III and Xba I enzyme; Be connected with the pUC19 carrier of cutting through Hind III and Xba I enzyme equally then, obtain inserting the recombinant vectors pUC19-19KD2 of reverse goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P;

(4) make up the recombinant vectors pUC19-19KD2-poly (A) that contains terminator poly (A)

With expression vector p3301 DNA is template, carries out pcr amplification with the terminator primer of sequence shown in SEQ ID NO:9 and the SEQ ID NO:10, obtains the terminator poly (A) of sequence shown in SEQ ID NO:11; The terminator poly (A) that further has corresponding restriction enzyme site with Xho I and Hind III double digestion; Be connected with the same recombinant vectors pUC19-19KD2 that obtains through the step (3) of Xho I and Hind III double digestion then, obtain containing the recombinant vectors pUC19-19KD2-poly (A) of terminator poly (A);

(5) structure contains endosperm specificity expression promoter P-zp22/6, the recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) of forward and reverse purpose fragment 19-KD-P and terminator poly (A)

The recombinant vectors pUC19-p22/6-22KD1 of step (2) structure and the recombinant vectors pUC19-19KD2-poly (A) of step (4) structure are used Xba I and Hind III double digestion respectively; The enzyme that will contain reverse purpose fragment 19-KD-P and terminator poly (A) is then cut product 19KD2-poly (A) and is connected on the recombinant vectors pUC19-p22/6-19KD1 that step (2) makes up; Obtain containing endosperm specificity expression promoter P-zp22/6, the recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) of forward and reverse goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P and terminator poly (A);

(6) structure of the connection of gus gene intron and zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD

With plasmid vector pGreen-0229 Backbone DNA is template, carries out pcr amplification with the gus gene intron primer of sequence shown in SEQ ID NO:12 and the SEQ ID NO:13, obtains the gus gene intron of sequence shown in SEQ ID NO:14; The gus gene intron that further has corresponding restriction enzyme site with Xba I single endonuclease digestion; Be connected with the recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) that phosphorylation processed steps (5) makes up behind Xba I single endonuclease digestion equally then; Obtain described zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD, the nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD is shown in SEQ ID NO:21.

RNAi vector construction strategy of the present invention is: the prolamine the corn lysine content is had important decisive action is an object; Adopt first segmentation to make up intermediate carrier; Again each intermediate carrier is connected; Insert the mode of intron at last, be about to specificity promoter and forward purpose fragment and be building up on the same skeleton carrier pUC19; On another pUC19, insert reverse purpose fragment and terminator poly (A) simultaneously; Again reverse purpose fragment and terminator poly (A) are inserted into and contain on promotor and the segmental recombinant vectors of forward purpose; Insert the gus gene intron at last and constitute RNAi carrier of the present invention; Because 22-KD and 19-KD alcohol soluble protein gene all are endosperm specificity expressions, so have cloned a specificity promoter among the present invention, insert then on the RNAi carrier.Described RNAi vector construction synoptic diagram is as shown in Figure 1; The advantage of said construction of carrier is:

(1) through adding the gus gene intron, improves the reticent efficient of RNAi carrier;

(2) conventional RNAi construction of carrier be by promotor, forward purpose fragment, one section not the order of expressed sequence, reverse purpose fragment, terminator poly (A) be connected on the skeleton carrier one by one; The structure of this RNAi carrier is connected to promotor and forward purpose fragment, reverse purpose fragment and terminator poly (A) on the skeleton carrier pUC19; And then two carriers are carried out enzyme cut and be connected, and then reverse purpose fragment and terminator poly (A) be inserted into contain on promotor and the segmental recombinant vectors of forward purpose.Because two intermediate carriers can make up simultaneously, therefore shortened the time of vector construction, improved efficient.

(3) the zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD and the pUC19-RNAi-19KD maize transformation kind that make up with the inventive method; Obtained high-lysine content transfer-gen plant; Compare with the non-transgenic plant, the seed lysine content of transgenic corns has improved 15-60%, compares with the high lysine corn varieties that existing traditional breeding method is cultivated; Lysine content has the raising of peer-level or the high lysine corn varieties that a little higher than traditional breeding method is cultivated; And (5-7) compares with conventional breeding, and the transformation cycle significantly shortens, and only needs 1-2.Simultaneously, the parallel laboratory test explanation has better repeatability and stability based on the corn gene technology of this RNAi carrier.The present invention has solved in the conventional breeding and has utilized through the contents level of the synthetic and then raising Methionin of RNA perturbation technique adjusting zein in addition O2The high lysine corn varieties endosperm of gene seed selection is soft and key issue such as disease resistance difference, has realized utilizing the target of the corn novel material of RNA interference principle initiative high-lysine content, and is significant for the corn quality improvement.

In the sequence table shown in the SEQ ID NO:1 is the upstream primer sequence of the specific promoter p primer of zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD of the present invention or RNAi carrier pUC19-RNAi-19KD.

In the sequence table shown in the SEQ ID NO:2 is the downstream primer sequence of the specific promoter p primer of zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD of the present invention or RNAi carrier pUC19-RNAi-19KD.

In the sequence table shown in the SEQ ID NO:3 is the nucleotide sequence of endosperm specificity expression promoter P-zp22/6.

In the sequence table shown in the SEQ ID NO:4 is the upstream primer sequence of the R22-6 primer of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P.

In the sequence table shown in the SEQ ID NO:5 is the downstream primer sequence of the R22-6 primer of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P.

In the sequence table shown in the SEQ ID NO:6 is the upstream primer sequence of the R22-7 primer of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P.

In the sequence table shown in the SEQ ID NO:7 is the downstream primer sequence of the R22-7 primer of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P.

In the sequence table shown in the SEQ ID NO:8 is the nucleotide sequence of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P.

In the sequence table shown in the SEQ ID NO:9 is the upstream primer sequence of terminator poly (A).

In the sequence table shown in the SEQ ID NO:10 is the downstream primer sequence of terminator poly (A).

In the sequence table shown in the SEQ ID NO:11 is the nucleotide sequence of terminator poly (A).

In the sequence table shown in the SEQ ID NO:12 is the upstream primer sequence of gus gene intron.

In the sequence table shown in the SEQ ID NO:13 is the downstream primer sequence of gus gene intron.

In the sequence table shown in the SEQ ID NO:14 is the nucleotide sequence of gus gene intron.

In the sequence table shown in the SEQ ID NO:15 is the nucleotide sequence of zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD of the present invention.

In the sequence table shown in the SEQ ID NO:16 is the upstream primer sequence of the R19-4 primer of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P.

In the sequence table shown in the SEQ ID NO:17 is the downstream primer sequence of the R19-4 primer of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P.

In the sequence table shown in the SEQ ID NO:18 is the upstream primer sequence of the R19-5 primer of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P.

In the sequence table shown in the SEQ ID NO:19 is the downstream primer sequence of the R19-5 primer of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P.

In the sequence table shown in the SEQ ID NO:20 is the nucleotide sequence of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P.

In the sequence table shown in the SEQ ID NO:21 is the nucleotide sequence of zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD of the present invention.

In the sequence table shown in the SEQ ID NO:22 is that RNAi carrier pUC19-RNAi-22KD transforms T ₀Detect the upstream primer sequence of 22Y3 primer for the PCR of regeneration plant.

In the sequence table shown in the SEQ ID NO:23 is that RNAi carrier pUC19-RNAi-22KD transforms T ₀Detect the downstream primer sequence of 22Y3 primer for the PCR of regeneration plant.

In the sequence table shown in the SEQ ID NO:24 is that RNAi carrier pUC19-RNAi-19KD transforms T ₀Detect the upstream primer sequence of 19Y5 primer for the PCR of regeneration plant.

In the sequence table shown in the SEQ ID NO:25 is that RNAi carrier pUC19-RNAi-19KD transforms T ₀Detect the downstream primer sequence of 19Y5 primer for the PCR of regeneration plant.

Description of drawings

Fig. 1 is the structure synoptic diagram of RNAi carrier pUC19-RNAi-22KD or pUC19-RNAi-19KD.

Fig. 2 is the PCR qualification result of endosperm specific expression promoter P-zp22/6,

Swimming lane M. DNA marker, 1. the PCR product of endosperm specific expression promoter P-zp22/6.

Fig. 3 is the PCR qualification result of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P,

Swimming lane M. DNA Marker, 1. the PCR product of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P.

Fig. 4 is the PCR qualification result of terminator poly (A),

Swimming lane M. DNA Marker, 1. the PCR product of terminator poly (A).

Fig. 5 is the PCR qualification result of gus gene intron,

Swimming lane M. DNA marker, 1. the PCR product of gus gene intron.

Fig. 6 is the PCR qualification result of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P,

Swimming lane M. DNA Marker, 1. the PCR product of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P.

Fig. 7 is that the enzyme of RNAi carrier pUC19-RNAi-22KD and pUC19-RNAi-19KD is cut detection,

The A:pUC19-RNAi-22KD restriction analysis, B: be the pUC19-RNAi-19KD restriction analysis.Swimming lane M. DNA Marker, 1 with 5:Xba I and Hind III double digestion, 2 with 6:Xba I single endonuclease digestion, 3 with 7:Sac I and Xba I double digestion, 4 and 8:Sac I and Hind III double digestion.

Fig. 8 is a pUC19-RNAi-22KD carrier collection of illustrative plates.

Fig. 9 is a pUC19-RNAi-19KD carrier collection of illustrative plates.

Figure 10 is the maize callus.

Figure 11 is to be the callus after example explanation zein spirit-soluble gene RNAi vector gene rifle transforms with the pUC19-RNAi-19KD carrier.

Figure 12 is to be the regeneration of callus after example explanation zein spirit-soluble gene RNAi vector gene rifle transforms with the pUC19-RNAi-19KD carrier.

Figure 13 is to be the strong seedling culture of differentiation seedling after example explanation zein spirit-soluble gene RNAi carrier transforms with the pUC19-RNAi-19KD carrier.

Figure 14 is to be that example explanation zein spirit-soluble gene RNAi carrier transforms the T that obtains with the pUC19-RNAi-19KD carrier ₀Field performance for transfer-gen plant.

Figure 15 is to be that example explanation zein spirit-soluble gene RNAi carrier transforms the T that obtains with the pUC19-RNAi-19KD carrier ₀Solid situation for transfer-gen plant.

Figure 16 is the T that the pUC19-RNAi-22KD carrier transforms ₀For the PCR evaluation of transfer-gen plant,

Swimming lane M. DNA Marker, CK+: positive control (amplification of pUC19-RNAi-22KD interference carrier), CK-: negative control (amplification of B73 non-transgenic plant), CK: blank (water is template), 1-9:T ₀Increase for transfer-gen plant.

Figure 17 is the T that the pUC19-RNAi-19KD carrier transforms ₀For the PCR evaluation of transfer-gen plant,

Swimming lane M. DNA Marker, CK+: positive control (amplification of pUC19-RNAi-19KD interference carrier), CK-: negative control (amplification of B73 non-transgenic plant), CK: blank (water is template), 1-7:T ₀Increase for transfer-gen plant.

Embodiment

All ingredients used in the experiment all can be bought from the commercial channel, and the skeleton carrier pUC19 that uses in the experiment, plasmid vector pGreen-0229 backbone, expression vector P3301, T carrier and plasmid extraction kit, glue reclaim test kit, Marker, high-fidelity DNA polymerase, dNTPs, bacillus coli DH 5 alpha competent cell and bar gene all available from the Beijing Quanshijin Biotechnology Co., Ltd; Restriction enzyme, T4-DNA ligase enzyme are available from U.S. NEB company; Reagent such as penbritin, IPTG, X-gal are all available from the precious biotechnology in Dalian ltd; RQ DNA enzyme, reversed transcriptive enzyme, 5 * reaction buffer, dNTP, RNA enzyme inhibitors are available from Promega company; Other chemical reagent are homemade analytical pure; Particle gun PDS-1000/He and running stores thereof are available from Bio-Rad company.Nucleotide sequencing is accomplished by the big genome company of China.Used primer sequence is synthetic by Beijing AudioCodes biotech company.Method therefor is ordinary method if no special instructions in the experiment.

Embodiment 1 zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD makes up

One, supply the examination material: corn ( Zea maysL.) self-mating system B73 is the corn germplasm that is widely used in maize genetic research and breeding practice at present, can from seeds company (like auspicious the kinds industry in field, Yunnan ltd, address: tap street, the north suburb, Kunming, Yunnan Province) directly purchase.

Two, experimental technique:

1, the extraction of B73 corn gene group DNA:

1) gets maize leaf 300 mg, add liquid nitrogen and fully grind;

2) the 2%CTAB extracting solution of 65 ℃ of preheatings of adding 700 μ l, mixing gently;

3) place 65 ℃ of water-bath 30-60 min;

4) room temperature is placed 2 min, adds 300 μ l chloroform-primary isoamyl alcohol (24:1), fully shakes 1-2 h;

5) centrifugal 10 min of 10,000 rpm;

6) get in upper strata water to the new centrifuge tube, add 600 μ l Virahols, room temperature is placed 10 min;

7) centrifugal 1 min of 10,000 rpm abandons supernatant;

8) the ethanol 500 μ l of adding 75%, room temperature is placed 30 min;

9) repeating step 7 and 8;

10) centrifugal 30 s of 10,000 rpm abandon supernatant, seasoning DNA;

11) add 100 μ l TE damping fluid dissolving DNAs;

12) detect back packing ,-70 ℃ of preservations.

2) reaction conditions: 95 ℃ of preparatory sex change 5 min, (72 ℃ are extended 1 min for 95 ℃ of sex change 50 s, 57 ℃ of annealing 50 s) 35 circulations, 72 ℃ are extended 10 min.The PCR product is electrophoresis detection on 1% sepharose.

3, the extraction of the total RNA of B73 corn kernel:

1) takes from the corn kernel 50-100 mg that hands over pollination back 18 days, fully grind with liquid nitrogen;

2) add 1 ml Trizol reagent, concuss mixing;

3) centrifugal 10 min of 12,000 rpm get supernatant;

4) add 200 μ l chloroforms, concuss mixing, ice bath 3 min;

5) centrifugal 15 min of 12,000 rpm (4 ℃);

6) get in upper strata water to the new centrifuge tube, add the Virahol of equivalent volumes, place 10 min for-20 ℃;

7) centrifugal 15 min of 12,000 rpm (4 ℃) abandon supernatant, add 700 μ l, 70% washing with alcohol deposition;

8) centrifugal 10 min of 8,000 rpm (4 ℃) abandon supernatant;

9) deposition is with 30 μ l DEPC-H ₂The O dissolving;

10) 1% agarose gel electrophoresis detects RNA.

4, RNA reverse transcription system

Reaction system: the total RNA 8 μ l of corn kernel, add 1 μ l RQ DNA enzyme and 1 μ l RQ damping fluid, 37 ℃ of water-bath 30 min, taking-up is placed on ice, adds 1 μ l RQ stop buffer, 65 ℃ of water-bath 10 min; Take out 9 μ l then as the RNA template, add OligodT 1 μ l, mixing is placed on 70 ℃ of water-bath 5 min, cooled on ice 2 min then; Add 5 times of RT MLV damping fluid 4 μ l again, 10 mM dNTP, 4 μ l, RNA enzyme inhibitors 1 μ l (20U), M-MLV ThermoScript II 1 μ l (200U), 42 ℃ of water-bath 1 h; 72 ℃ of deactivation 10 min, ice bath 10 min are in-20 ℃ of preservations.

Reaction conditions: 94 ℃ of preparatory sex change 5 min, (72 ℃ are extended 1 min for 94 ℃ of sex change 40 s, 59 ℃ of annealing 45 s) 35 circulations, 72 ℃ are extended 10 min.The PCR product is electrophoresis detection on 1% sepharose.

7, connect product to colibacillary conversion

1) gets competent cell DH5 α 100 μ l, add T carrier connector 2 μ l, ice bath 30 min;

2) 42 ℃ of water-bath heat shock 90 s place 2 min on ice immediately;

3) add 500 μ l LB substratum, 200 * g shakes 1 h under 37 ℃ of conditions;

4) centrifugal 1 min of 3500 * g abandons the part supernatant after leaving standstill 1 h;

5) add 5 μ l ZPTG and 50 μ l X-Gal;

6) coated plate, seal;

7) cultivate 16 h for 37 ℃;

8) choose white single bacterium colony, 37 ℃ are shaken bacterium.

8, the extraction of recombinant vectors DNA:

Extract with Beijing Quanshijin Biotechnology Co., Ltd's plasmid extraction kit, step is following:

1) gets centrifugal 1 min of bacterium 10,000 * g of 4 ml incubated overnight;

2) add the colourless RB of 250 μ l (containing RNase A), concussion suspension bacterial precipitation;

3) add the blue LB of 250 μ l, be no more than 5 min;

4) add the yellow NB of 350 μ l, room temperature leaves standstill 2 min;

5) centrifugal 5 min of 15,000 * g get supernatant and add in the adsorption column;

6) centrifugal 1 min of 15,000 * g, the abandoned stream fluid;

7) add 650 μ l solution W B, centrifugal 1 min of 15,000 * g, abandoned stream fluid;

8) centrifugal 2 min of 15,000 * g thoroughly remove residual WB;

9) adsorption column is placed clean centrifuge tube, EB or deionized water (pH>7.0) room temperature that in post, adds 70 ℃ of preheatings of 50 μ l leaves standstill 1 min;

10) centrifugal 1 min of 10,000 * g, eluted dna is in-20 ℃ of preservations.

10, endonuclease bamhi reclaims and is connected

Reclaim test kit (Beijing Quanshijin Biotechnology Co., Ltd) with glue and reclaim endonuclease bamhi, the concrete operations step is following:

1) cuts target DNA band in the sepharose, weigh;

2) add 3 times of volume solution GSB, in 55 ℃ of water-bath 10 min;

3) treat that gelating soln reduces to room temperature, add in the adsorption column and leave standstill 1 min, centrifugal 1 min of 10,000 * g, abandoned stream fluid;

4) add 650 μ l solution W B, centrifugal 1 min of 10,000 * g, abandoned stream fluid;

5) centrifugal 2 min of 10,000 * g remove residual WB;

6) adsorption column is placed centrifuge tube, in post, add 70 ℃ of preheating EB of 50 μ l or deionized water, room temperature leaves standstill 1 min;

7) centrifugal 1 min of 10,000 * g, eluted dna;

Reaction conditions: mix back 65 ℃ of water-bath 5 min gently, place 1 min on ice immediately, add 1.5 μ l then and connect damping fluid and 0.5 μ l T4 ligase enzyme, 16 ℃ of connections of spending the night.

11, the evaluation of recombinant vectors

1) PCR identifies

Reaction system and condition see two, 2; According to the PCR detected result, get the positive colony adding and contain antibiotic LB liquid nutrient medium, 200 * g shakes 1 h under 37 ℃ of conditions; Get 1 ml bacterium liquid then and send the order-checking of Beijing China big gene biological technology Ltd, sequencing result carries out sequential analysis with seqMan software; Target fragment to transformed into escherichia coli in ncbi database is carried out the homology comparison with the sequence of zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD or pUC19-RNAi-19KD respectively.

2) enzyme is cut evaluation

Method is with embodiment 1 two steps 9.

Below each step genome and the recombinant vectors DNA that relate to corn variety B73 carry out pcr amplification; The total RNA of B73 corn kernel is carried out the RT-PCR amplification; The T carrier of PCR product connects, and the ligation system and the reaction conditions that make up each carrier are all undertaken by the aforesaid operations step.

Three, the acquisition of endosperm specificity expression promoter P-zp22/6

1, the p primer of design endosperm specificity expression promoter P-zp22/6

According to (A new opaque variant of maize by a single dominant RNA-interference-inducing transgene. Genetics 2003 such as Segal; 165:387-397) report, P-zp22/6 is the promotor of endosperm specificity expression gene zp22/6.The mRNA of zp22/6 gene is at first found in this experiment in MaizeGDB DB (http://www.maizegdb.org/); With this mRNA is that motif finds corresponding zp22/6 genomic dna sequence and respective coding district in Genbank (accession number is AC144719.1), and the sequence of preceding 1.3 kb in coding region is the promotor of this gene.Select the long sequence of 1.5 kb before the coding region as target sequence for finding complete promotor; And according to the MCS analysis that exists on the pUC19 carrier; Utilize Sac I and Xba I can endosperm specificity expression promoter P-zp22/6 be connected to the pUC19 carrier, therefore with the Primer5 software design primer sequence (abbreviation specific promoter p primer) of P-zp22/6 of following band restriction enzyme site:

2, the acquisition of endosperm specific expression promoter P-zp22/6 sequence

With the B73 corn gene group DNA is template; With SEQ ID NO:1 (upstream primer) and SEQ ID NO:2 (downstream primer) is that primer is right; Carry out pcr amplification (amplification is seen Fig. 2), obtain the endosperm specificity expression promoter P-zp22/6 fragment that the 5' end is introduced Sac I restriction enzyme site, 3' end introducing Xba I and Sma I restriction enzyme site; Then this P-zp22/6 fragment is connected with the T carrier and to its order-checking, sequencing result show 5' end and 3' end the restriction enzyme site of introducing really be Sac I restriction enzyme site, Xba I and Sma I restriction enzyme site;

3, checking: in the NCBI website (on the http://www.ncbi.nlm.nih.gov/BALST; Accession number is AC144719.1) to the checking of comparing of step 3,2 sequencing result; Discovery is introduced the restriction enzyme site except that 5' end, 3' end; The similarity of corresponding sequence reaches 100% among sequencing result and the GeneBank, shows that institute's cloned sequence really is endosperm specific expression promoter P-zp22/6 sequence.Said endosperm specific expression promoter P-zp22/6 sequence is shown in SEQ ID NO:3.

Four, make up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6

Step 3 P-zp22/6 fragment is connected the order-checking plasmid that obtains cuts with Sac I and Xba I enzyme with the T carrier; Be connected with the carrier pUC19 that cuts through Sac I and Xba I enzyme equally then, obtain containing the recombinant vectors pUC19-p22/6 of endosperm specificity expression promoter zp22/6.

Five, the acquisition of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

1, the primer of design 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

According to (A new opaque variant of maize by a single dominant RNA-interference-inducing transgene. Genetics 2003 such as Segal; The MCS analysis of the 22-KD primer sequence of the 22-KD alcohol soluble protein gene of 165:387-397) delivering and recombinant vectors pUC19-p22/6 and skeleton carrier pUC19; Utilize Sma I and Xba I can the forward fragment of goal gene be connected to the pUC19-p22/6 carrier; And utilize Hind III and Xba I can the reverse fragment of goal gene be connected to the pUC19 carrier, therefore designed the primer sequence (being called for short R22-6 primer and R22-7 primer) of the 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P of following band restriction enzyme site:

2, the acquisition of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

With the total RNA of B73 corn kernel is template, is that primer is right with SEQ ID NO:4 (upstream primer) and SEQ ID NO:5 (downstream primer), carries out the RT-PCR amplification, obtains holding the 22-KD-P fragment of introducing Sma I and Xba I restriction enzyme site respectively at 5' end and 3'; With this fragment is template; With SEQ ID NO:6 (upstream primer) and SEQ ID NO:7 (downstream primer) is that primer is right; Carry out RT-PCR amplification (amplification is seen Fig. 3), obtain further having introduced the 22-KD-P fragment about 300 bp of Hind III and Xho I restriction enzyme site at the 3' end; This 22-KD-P fragment of introducing restriction enzyme site is connected to T carrier and order-checking, and sequencing result shows successfully introduces Hind III and Xho I restriction enzyme site at 22-KD-P fragment 3' end, introduces Sma I and Xba I restriction enzyme site at the 5' end;

3, checking: in the NCBI website (on the http://www.ncbi.nlm.nih.gov/BALST; Accession number is FL392908.1) the segmental sequencing result of this 22-KD-P is compared; Discovery is introduced the above-mentioned restriction enzyme site except that 5' end, 3' end; The similarity of corresponding sequence reaches 99% among sequencing result and the GeneBank, proves that this sequence really is the 22-KD-P fragment of 22-KD alcohol soluble protein gene, can be used for the RNAi vector construction.The 22-KD-P fragment sequence of said 22-KD alcohol soluble protein gene is shown in SEQ ID NO:8.

Six, forward inserts the 22-KD-P fragment in containing the recombinant vectors pUC19-p22/6 of endosperm specificity expression promoter P-zp22/6

Step 5 22-KD-P fragment is connected the order-checking plasmid that obtains cuts with Sma I and Xba I enzyme with the T carrier; Be connected with the pUC19-p22/6 recombinant vectors that the step 4 of cutting through Sma I and Xba I enzyme equally obtains then, obtain containing endosperm specificity expression promoter P-zp22/6 and forward and insert the segmental recombinant vectors pUC19-p22/6-22KD1 of 22-KD-P.

Seven, make up the segmental recombinant vectors pUC19-22KD2 of the reverse 22-KD-P of insertion

Step 5 22-KD-P fragment is connected the order-checking plasmid that obtains cuts with the T carrier, be connected with the pUC19 carrier of cutting through Hind III and Xba I enzyme equally then, oppositely inserted the segmental recombinant vectors pUC19-22KD2 of 22-KD-P with Hind III and Xba I enzyme.

Eight, make up the recombinant vectors pUC19-22KD2-poly (A) that contains terminator poly (A)

1, the primer of design terminator poly (A)

Terminator sequence (http://www.cambia.org/daisy/cambia/home.html) and recombinant vectors pUC19-22KD2 according to expression vector p3301 go up the MCS analysis that exists; Utilize Xho I and Hind III can terminator poly (A) be connected to the pUC19-22KD2 carrier, therefore with the Primer5 software design terminator poly (A) primer sequence of following band restriction enzyme site:

2, the acquisition of terminator poly (A)

With the p3301 expression vector dna is template; With SEQ ID NO:9 (upstream primer) and SEQ ID NO:10 (downstream primer) is that primer is right; Carry out pcr amplification (amplification is seen Fig. 4), obtain the terminator poly (A) that the 5' end is introduced Xho I restriction enzyme site, 3' end introducing Hind III restriction enzyme site; This fragment is connected to T carrier and order-checking, sequencing result show 5' end and 3' end the restriction enzyme site of introducing really be Xho I and Hind III restriction enzyme site;

3, checking: corresponding sequence among the sequencing result of terminator poly (A) and the p3301 is compared, and the two similarity reaches 100%, proves that institute's calling sequence is the terminator sequence in the p3301 expression vector.Said terminator poly (A) sequence is shown in SEQ ID NO:11.

4, step 82 terminator poly (A) being connected the order-checking plasmid that obtains with the T carrier cuts with Xho I and Hind III enzyme; Be connected the recombinant vectors pUC19-22KD2-poly (A) that obtains inserting reverse insertion 22-KD-P fragment and contain terminator poly (A) then with the recombinant vectors pUC19-22KD2 that the step 7 of cutting through Xho I and Hind III enzyme equally obtains.

Nine, structure contains endosperm specificity expression promoter P-zp22/6, the recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) of forward 22-KD-P fragment, reverse 22-KD-P fragment and terminator poly (A)

The recombinant vectors pUC19-22KD2-poly (A) that recombinant vectors pUC19-p22/6-22KD1 that step 6 is obtained and step 8 obtain uses Xba I and Hind III double digestion respectively; The enzyme that will contain reverse 22-KD-P fragment and terminator poly (A) is then cut product 22KD2-poly (A) and is connected to recombinant vectors pUC19-p22/6-22KD1, obtains containing the recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) of endosperm specificity expression promoter P-zp22/6, forward 22-KD-P fragment, reverse 22-KD-P fragment and terminator poly (A).

Ten, the acquisition of gus gene intron

1, the primer of design gus gene intron

Gus gene sequence (http://www.pgreen.ac.uk/JIT/pG0229.htm) and recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) according to pGreen-0229 Backbone carrier go up the MCS analysis that exists; Utilize Xba I restriction enzyme site can the gus gene intron be connected to pUC19-p22/6-22KD1-22KD2-poly (A) carrier, therefore with the Primer5 software design primer sequence of gus gene intron of following band restriction enzyme site:

2, the acquisition of gus gene intron

With pGreen-0229 Backbone carrier DNA is template; With SEQ ID NO:12 (upstream primer) and SEQ ID NO:13 (downstream primer) is that primer is right; Carry out pcr amplification (amplification is seen Fig. 5), obtain the gus gene intron that 5' and 3' end is all introduced Xba I restriction enzyme site; This fragment is connected to T carrier and order-checking, sequencing result show 5' end and 3' end the restriction enzyme site of introducing really be Xba I restriction enzyme site;

3, checking: the corresponding sequence to pGreen-0229 Backbone carrier on the sequencing result of gus gene intron and the http://www.pgreen.ac.uk/JIT/pG0229.htm website is compared; Find that the two similarity reaches 100%, prove that institute's calling sequence is the gus gene intron sequences.Said gus gene intron sequences is shown in SEQ ID NO:14;

11, make up zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD of the present invention

1, makes up zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD

Step 10 gus gene intron is connected the order-checking plasmid that obtains with Xba I single endonuclease digestion with the T carrier; Be connected with the same recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) that phosphorylation is handled behind Xba I single endonuclease digestion then, obtain zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD of the present invention;

2, the checking of zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD

The bacterium liquid that will contain zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD is delivered to the big genome company of China and is carried out the nucleotide sequence order-checking; Sequencing result shows and has successfully made up zein spirit-soluble gene RNAi carrier, called after pUC19-RNAi-22KD; Simultaneously according to the vector construction flow process, carry out enzyme with the corresponding enzyme of restriction enzyme site and cut detection (shown in Fig. 7 A) result and show that the pUC19-RNAi-22KD vector construction finishes.The nucleotide sequence of said zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD shown in SEQ ID NO:15, structure is as shown in Figure 8.

Through between the restriction enzyme site Sac I of skeleton carrier pUC19 and Sma I, inserting endosperm specific expression promoter P-zp22/6; Forward inserts purpose fragment 22-KD-P between Sma I and Xba I restriction enzyme site; The reverse purpose fragment 22-KD-P that inserts between restriction enzyme site Xba I and Xho I; Between forward 22-KD-P fragment and reverse 22-KD-P fragment, insert the gus gene intron simultaneously, between restriction enzyme site Hind III and Xho I, insert terminator poly (A), successfully made up the described zein spirit-soluble gene RNAi of present embodiment carrier pUC19-RNAi-22KD; The nucleotide sequence of described endosperm specific expression promoter P-zp22/6 is shown in SEQ ID NO:3; The nucleotide sequence of described 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P is shown in SEQ ID NO:8; Shown in the nucleotide sequence SEQ ID NO:14 of described gus gene intron; Shown in the nucleotide sequence SEQ ID NO:11 of described terminator poly (A), the nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD shown in SEQ ID NO:15, collection of illustrative plates is as shown in Figure 8.

The structure of embodiment 2, zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD

The structure of said zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD is except that following step difference, and all the other steps are identical with embodiment 1, repeat no more.

One, the acquisition of endosperm specificity expression promoter P-zp22/6, method is identical with embodiment 1.

Two, make up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6, method is identical with embodiment 1.

Three, the acquisition of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

1, the primer of design 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

In DB MaizeGDB, find the mRNA sequence of 19-KD gene; With this mRNA is that motif is gone up the 19-KD genomic dna sequence that correspondence is found in comparison at Genebank (accession number is AC196717.3); MCS analysis to this sequence and recombinant vectors pUC19-p22/6 and skeleton carrier pUC19; Utilize Sma I and Xba I can the forward fragment of goal gene be connected to the pUC19-p22/6 carrier; And utilize Hind III and Xba I can the reverse fragment of goal gene be connected to the pUC19 carrier, therefore designed the primer (being called for short R19-4 primer and R19-5 primer) of the 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P of following band restriction enzyme site:

2, the acquisition of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

With the total RNA of B73 corn kernel is template; With SEQ ID NO:16 (upstream primer) and SEQ ID NO:17 (downstream primer) is that primer is right; Carry out the RT-PCR amplification, obtain holding the 19-KD-P fragment of introducing Sma I and Xba I restriction enzyme site respectively at 5' end and 3'; With this fragment is template; With SEQ ID NO:18 (upstream primer) and SEQ ID NO:19 (downstream primer) is that primer is right; Carry out RT-PCR amplification (amplification is seen Fig. 6), obtain further having introduced the 19-KD-P fragment about 300 bp of Hind III and Xho I restriction enzyme site at the 3' end; This 19-KD-P fragment of introducing restriction enzyme site is connected to T carrier and order-checking, and sequencing result shows successfully introduces Hind III and Xho I restriction enzyme site at 19-KD-P fragment 3' end, introduces Sma I and Xba I restriction enzyme site at the 5' end;

3, checking: in the NCBI website (on the http://www.ncbi.nlm.nih.gov/BALST; Accession number is FM187060.1) the segmental sequencing result of this 19-KD-P is compared; Discovery is introduced the restriction enzyme site except that 5' end, 3' end; The similarity of corresponding sequence reaches 99% among sequencing result and the GeneBank, proves that this sequence really is the 19-KD-P fragment of 19-KD alcohol soluble protein gene, can be used for the RNAi vector construction.The 19-KD-P fragment sequence of said 19-KD alcohol soluble protein gene is shown in SEQ ID NO:20.

Four, forward inserts the 19-KD-P fragment in containing the recombinant vectors pUC19-p22/6 of endosperm specificity expression promoter P-zp22/6

Present embodiment step 3 19-KD-P fragment is connected the order-checking plasmid that obtains cuts with Sma I and Xba I enzyme with the T carrier; Be connected with the pUC19-p22/6 recombinant vectors that the present embodiment step 2 of cutting through Sma I and Xba I enzyme equally obtains then, obtain containing endosperm specificity expression promoter P-zp22/6 and forward and insert the segmental recombinant vectors pUC19-p22/6-19KD1 of 19-KD-P.

Five, make up the segmental recombinant vectors pUC19-19KD2 of the reverse 19-KD-P of insertion

Present embodiment step 3 19-KD-P fragment is connected the order-checking plasmid that obtains cuts with Hind III and Xba I enzyme with the T carrier; Be connected with the pUC19 carrier of cutting through Hind III and Xba I enzyme equally then, oppositely inserted the segmental recombinant vectors pUC19-19KD2 of 19-KD-P.

Six, make up the recombinant vectors pUC19-19KD2-poly (A) that contains terminator poly (A)

1, the design of primers of terminator poly (A), acquisition and the checking of terminator poly (A), method is all identical with embodiment 1;

2, present embodiment step 61 terminator poly (A) being connected the order-checking plasmid that obtains with the T carrier cuts with Xho I and Hind III enzyme; Be connected the recombinant vectors pUC19-19KD2-poly (A) that obtains inserting reverse insertion 19-KD-P fragment and contain terminator poly (A) then with the recombinant vectors pUC19-19KD2 that the present embodiment step 5 of cutting through Xho I and Hind III enzyme equally obtains.

Seven, structure contains endosperm specificity expression promoter P-zp22/6, the recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) of forward 19-KD-P fragment, reverse 19-KD-P fragment and terminator poly (A)

The recombinant vectors pUC19-19KD2-poly (A) that recombinant vectors pUC19-p22/6-19KD1 that the present embodiment step 4 is obtained and step 6 obtain uses Xba I and Hind III double digestion respectively; To then the enzyme that contains reverse 19-KD-P fragment and terminator poly (A) be cut product 19KD2-poly (A) then and be connected to recombinant vectors pUC19-p22/6-19KD1, obtain containing the recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) of endosperm specificity expression promoter P-zp22/6, forward 19-KD-P fragment, reverse 19-KD-P fragment and terminator poly (A).

Eight, the acquisition of gus gene intron, method is identical with embodiment 1.

Nine, make up zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD of the present invention

1, makes up zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD

Present embodiment step 9 gus gene intron is connected the order-checking plasmid that obtains with Xba I single endonuclease digestion with the T carrier; Be connected with the same recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) that phosphorylation is handled behind Xba I single endonuclease digestion then, obtain zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD of the present invention;

2, the checking of zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD

The bacterium liquid that will contain zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD is delivered to the big genome company of China and is carried out the nucleotide sequence order-checking; Sequencing result shows and has successfully made up zein spirit-soluble gene RNAi carrier, called after pUC19-RNAi-19KD; Simultaneously according to the vector construction flow process, carry out enzyme with the corresponding enzyme of restriction enzyme site and cut detection (shown in Fig. 7 B) result and show that the pUC19-RNAi-19KD vector construction finishes.The nucleotide sequence of said zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD shown in SEQ ID NO:21, structure is as shown in Figure 9.

Through between the restriction enzyme site Sac I of skeleton carrier pUC19 and Sma I, inserting endosperm specific expression promoter P-zp22/6; Forward inserts purpose fragment 19-KD-P between Sma I and Xba I restriction enzyme site; The reverse purpose fragment 19-KD-P that inserts between restriction enzyme site Xba I and Xho I; Between forward 19-KD-P fragment and reverse 19-KD-P fragment, insert the gus gene intron simultaneously, between restriction enzyme site Hind III and Xho I, insert terminator poly (A), successfully made up the described zein spirit-soluble gene RNAi of present embodiment carrier pUC19-RNAi-19KD; The nucleotide sequence of described endosperm specific expression promoter P-zp22/6 is shown in SEQ ID NO:3; The nucleotide sequence of described 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P is shown in SEQ ID NO:20; Shown in the nucleotide sequence SEQ ID NO:14 of described gus gene intron, shown in the nucleotide sequence SEQ ID NO:11 of described terminator poly (A); The nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD shown in SEQ ID NO:21, collection of illustrative plates is as shown in Figure 9.

Using aforesaid method to make up the RNAi carrier of the present invention that obtains is to be object with the prolamine that the corn lysine content is had important decisive action; Adopt first segmentation to make up intermediate carrier; Again each intermediate carrier is connected; Insert the mode of intron at last, be about to specificity promoter and forward purpose fragment and be building up on the same skeleton carrier pUC19; On another pUC19, insert reverse purpose fragment and terminator poly (A) simultaneously; Again reverse purpose fragment and terminator poly (A) are inserted into and contain on promotor and the segmental recombinant vectors of forward purpose; Insert the gus gene intron at last and constitute RNAi carrier of the present invention; Because 22-KD and 19-KD alcohol soluble protein gene all are endosperm specificity expressions, so have cloned a specificity promoter among the present invention, insert then on the RNAi carrier.Its advantage of RNAi carrier that makes up with this method mainly comprises: 1) through adding the gus gene intron, improve the reticent efficient of RNAi carrier; 2) conventional RNAi construction of carrier be by promotor, forward purpose fragment, one section not the order of expressed sequence, reverse purpose fragment, terminator poly (A) be connected on the skeleton carrier one by one; The structure of this RNAi carrier is connected to promotor and forward purpose fragment, reverse purpose fragment and terminator poly (A) on the skeleton carrier pUC19; And then two carriers are carried out enzyme cut and be connected, and then reverse purpose fragment and terminator poly (A) be inserted into contain on promotor and the segmental recombinant vectors of forward purpose.Because two intermediate carriers can make up simultaneously, therefore shortened the time of vector construction, improved efficient.

The application of the zein spirit-soluble gene RNAi carrier that embodiment 3 the inventive method make up in improving the corn variety lysine content

Use zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD or pUC19-RNAi-19KD difference maize transformation kind that the present invention makes up, all make its seed lysine content level obtain bigger raising, reached the purpose of improvement corn quality.

One, supplies the examination material

Corn ( Zea maysL.) cross-fertilize seed H99 * Hi II B (purchasing the greatly healthy scientific and technological development of middle peasant ltd) in Beijing.18 d after the artificial autocopulation pollinates get fruit ear and sterilize the rataria of the about 10-1.2 mm of picking length under aseptic condition with 70% ethanol, 2.5% Youxiaolin and aqua sterilisa successively.

Two, substratum

It is as shown in table 1 that the inducing of maize callus, subculture, particle gun transform, transform used substratum such as screening and the regeneration plant differentiation of back callus, in 121 ℃ of autoclaving 20 min.

Annotate: macroelement, trace element, molysite, organic prescription by the N6 basic recipe prepare (Zhu Zhiqing etc. set up a kind of substratum of Rice Anther preferably through the nitrogenous source comparison test. Chinese science 1975, (5): 484-490), PH transfers to 5.8.

Three, rataria is cultivated

1, rataria is placed on the culture medium A (table 1), the dark cultivation 3-4 week forms callus under 27-28 ℃ of condition, and be shown in figure 10;

2, get that growing way is good, tissue looseness, faint yellow, callus that embryo property is good, succeeding transfer culture under 27-28 ℃ of condition, every 2-3 week subculture once, the acceptor material that the gained callus transforms as particle gun;

3,, be cut into 2 mm size and transfer to substratum B (table 1) callus upward for use at preceding 4 h of particle gun bombardment.

Four, particle gun transforms

1, the preparation of the little bullet of particle gun and conversion

1) according to Klein etc. (High velocity microprojectiles for delivering nucleic acids into living cells. Nature 1987, method 327:70-73) prepares bronze suspension-s (bronze diameter 1.0 μ m);

2) get bronze suspension-s (60 mg/ml) the 33 μ l of 4 ℃ of preservations, add 4 μ l DNAs (1 μ g/ μ l) successively, 50 μ l CaCl ₂(2.5 mol/L), 20 μ l spermidines (0.1 mol/L), 5 min that vibrate, room temperature leaves standstill 10 min, and DNA is deposited on the particulate carrier;

3) centrifugal 3 s of 12,000 rpm abandon supernatant; Add 200 μ l, 100% ethanol rinsing deposition, centrifugal 3 s of 12,000 rpm; Abandon supernatant, add 50 μ l, 100% ethanol, the low speed concussion keeps suspended state; Every rifle is got the DNA bronze suspension-s that 12.5 μ l prepare and is evenly coated on little missile-borne body, dries subsequent use;

4) utilizing Sac I and Hind III to scale off the back to the interference goal gene that makes up enzyme on pUC19-RNAi-22KD or the pUC19-RNAi-19KD carrier collects; Press the mixed of 3:1 then with the bar gene; Adopt the method for particle gun bombardment to be transferred to goal gene in the acceptor, the transgenic event statistics is seen table 2.

2, the screening of callus and plant regeneration

1) after the particle gun bombardment, the callus that particle gun is transformed places on the substratum B, and dark overnight cultures under 28 ℃ of conditions is transferred on the culture medium A 28 ℃ then and recovered to cultivate 5-7 days;

2) with on the callus transport medium C, succeeding transfer culture under 28 ℃ of conditions, 2 all subcultures are once selected normal, the WD callus of color and luster (shown in figure 11) succeeding transfer culture, subculture 3-4 time;

3) callus is transferred on the division culture medium D, under 16 h illumination/8 h dark conditions, under 28 ℃ of conditions, cultivated for 2 weeks, have green point to occur;

4) when the bud of callus differentiation grows to about 1-2 cm (shown in figure 12), be transferred to and continue cultivation among the substratum E;

5) select the seedling (shown in figure 13) of well developed root system in the 3-4 leaf phase, with warm water flush away substratum, transplanting is buried in the greenhouse, is transplanted to field planting after 2 weeks, as until blossom and bear fruit (shown in figure 14).

Five, T ₀PCR for regeneration plant detects

1, design T ₀Detect primer for plant PCR

Classify target sequence as with the nucleotides sequence of the RNAi carrier pUC19-RNAi-19KD shown in zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD shown in the SEQ ID NO:15 and the SEQ ID NO:21 respectively, utilize Primer5 software to design the T of sequence shown in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and the SEQ ID NO:25 respectively ₀Detect the 22Y3 primer to right for plant PCR with the 19Y5 primer;

2, pUC19-RNAi-22KD carrier T ₀PCR for regeneration plant detects

Extract the pUC19-RNAi-22KD carrier and transform the T that obtains ₀Genomic dna (method is with embodiment 1, two, 1) for regeneration plant (90); Extract the DNA (method is with embodiment 1, two, 9) of pUC19-RNAi-22KD carrier; Respectively with the DNA (CK+) of transfer-gen plant seed DNA, pUC19-RNAi-22KD carrier, corn variety B73 non-transgenic plant seed DNA (CK-), blank water (CK) as template; To carrying out pcr amplification (shown in figure 16), the result shows 90 T that obtained by the pUC19-RNAi-22KD carrier with the 22Y3 primer of sequence shown in SEQ ID NO:22 (22Y3 upstream primer) and the SEQ ID NO:23 (22Y3 downstream primer) ₀In plant, there are 10 strains to amplify the purpose band (comprising goal gene and intron sequences) of 1 kb, positive rate is 11.1%, explains that the 22KD alcohol soluble protein gene has been integrated into the corn gene group.

3, pUC19-RNAi-19KD carrier T ₀PCR for regeneration plant detects

Extract the pUC19-RNAi-19KD carrier and transform the T that obtains ₀Genomic dna (method is with embodiment 1, two, 1) for regeneration plant (120); Extract the DNA (method is with embodiment 1, two, 9) of pUC19-RNAi-19KD carrier; Respectively with the DNA (CK+) of transfer-gen plant seed DNA, pUC19-RNAi-19KD carrier, corn variety B73 non-transgenic plant seed DNA (CK-), blank water (CK) as template; To carrying out pcr amplification (shown in figure 17), the result shows 120 T that obtained by the pUC19-RNAi-19KD carrier with the 19Y5 primer of sequence shown in SEQ ID NO:24 (19Y5 upstream primer) and the SEQ ID NO:25 (19Y5 downstream primer) ₀In plant, there are 12 strains to amplify the purpose band (comprising goal gene and intron sequences) about 800 bp, positive rate is 10.0%, explains that the 19KD alcohol soluble protein gene has been integrated into the corn gene group.

Six, the Methionin acid content of transfer-gen plant is measured

Results transgenic T ₀Seed (shown in figure 15) for plant; Send agricultural product quality supervision and inspection center of the Ministry of Agriculture (Kunming) to analyze; According to the standard method at international corn wheat improvement center (CIMMYT), analyzed T with Perten-DA7200 near infrared grain quality analyser ₀T for plant ₁For the lysine content of seed, the result shows:

Transform in 10 transgenic corns materials that obtain with zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD of the present invention; There is 1 strain to improve about 52% than the lysine content of non-transgenic plant; Have 3 strain lysine contents to improve 15-20%, other 6 strains do not have considerable change.

In 12 transgenic lines that zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD maize transformation kind of the present invention obtains; There are 2 strains to improve 50-60% than the lysine content of non-transgenic plant; Have 4 strain lysine contents to improve 15-20%, other 7 strains do not have considerable change.

Above-mentioned experiment shows; Zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD or pUC19-RNAi-19KD maize transformation kind with the inventive method structure; Obtained high-lysine content transfer-gen plant; Compare with the non-transgenic plant; The seed lysine content of transgenic corns has improved 15-60%, compares with the high lysine corn varieties that existing traditional breeding method is cultivated, and lysine content has the raising of peer-level or the high lysine corn varieties that a little higher than traditional breeding method is cultivated.Simultaneously, the parallel laboratory test explanation has better repeatability and stability based on the corn gene technology of this RNAi carrier.The present invention has solved in the conventional breeding and has utilized through the contents level of the synthetic and then raising Methionin of RNA perturbation technique adjusting zein in addition O2The high lysine corn varieties endosperm of gene seed selection is soft and key issue such as disease resistance difference.

< 110>Cereal Crops Inst., Yunnan Prov. Agricultural Academy

Field, Yunnan auspicious kind industry ltd

 

< 120>a kind of zein spirit-soluble gene RNAi construction of carrier

 

<130> -

 

<160> 25

 

<170> PatentIn?version?3.3

 

<210> 1

<211> 26

<212> DNA

< 213>artificial sequence

 

<220>

< 223>upstream primer of specific promoter p primer

 

<400> 1

gagctcggca?aagggactgg?caaagg 26

 

<210> 2

<211> 37

<212> DNA

< 213>artificial sequence

<220>

< 223>downstream primer of specific promoter p primer

 

<400> 2

tctagactag?cccgggcgct?tcgcttggtg?tgtttct 37

 

<210> 3

<211> 1367

<212> DNA

< 213>corn (Zea mays L.)

 

<400> 3

ggcaaaggga?ctggcaaagg?ggcccactag?agggttcttt?gccgagtgcc?agttcaccag 60

acactcggca?aataaacctc?ctttgccgag?tgtcgccgtg?ggcactcggc?gaaggatccg 120

tcaccgtcac?ttggcgccgt?gacggtgact?tttctttgcc?gagtgccaaa?tggcactcgg 180

caaagtcttt?gccgagtgcc?cgacaaaaag?tactcggcaa?agatgctgtt?gccgatgtac 240

agttcgccga?gcgttctttg?ccgagtgtta?cactcggcaa?agcatttgcc?gagtgtaaaa 300

tagcctttgc?cgtgtgtctc?agacacacgg?caaagaagct?gattccggta?gtgtttgggg 360

ggcgactaac?acaaatcgat?gacctttgat?gaagatggat?gtatttctcc?cacaaaatct 420

tggatccatc?ctctactatg?aaaagtcatg?aattcacaag?acgttttcga?aaacaatttc 480

agcttcactt?gtaaagttgc?ttgtgcggct?gaagccaaga?aaacattaga?ggacaaacaa 540

taacttacta?tgcttttcgt?gaactgtttc?agaccagcag?taataattat?tggtcctaca 600

tgcattaatg?gataagcggc?tagcatgcca?agacggagca?ttagttagga?gataagacct 660

agcgtttcat?tgcatcccca?tgaaaggaat?actatcacta?ctgcatgaat?aattttcaaa 720

tgtattaaaa?actgatattg?aaatgtaggt?aatctgtccg?cctatgtgaa?atgtttaaca 780

gaggcagcca?ctctaggatg?ttgcctctat?taattactca?tagatgcagc?catccaactc 840

taggatttca?attagtctca?atctatgcat?tcgtctatgc?attctaggat?tacaattagt 900

ctcaatcttg?tagtatttgt?tccttccttg?tcaaatcact?tctcatctaa?ctactatgct 960

tgtttaacca?tcagaacata?actacaacaa?catccattta?taaaggcttc?gatagcaaac 1020

tttacatatt?catatcatgt?taaggttgtc?acatgtgtaa?aggtgaagag?atcatgcatg 1080

tcattccacg?tagataaaaa?gaatgcctat?ataaaaatgg?cacattttct?tgtaggtagt 1140

ggaaagtatc?tttccagcaa?agaccatata?atccgataaa?gctgataact?aaatgtcgaa 1200

atcgagtaga?tgccatatca?tctatacctt?atctgttgtt?tggaaaaaaa?aaagacaaaa 1260

tccaaaaaaa?aatataagag?atctcaccta?tataaatagc?tctgaaatca?gtagttaatc 1320

catcacccat?aatattttga?gcattcagaa?acacaccaag?cgaagcg 1367

 

<210> 4

<211> 26

<212> DNA

< 213>artificial sequence

 

<220>

< 223>upstream primer of the R22-6 primer of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

 

<400> 4

cccgggggcg?agcgtctaca?acaacc 26

 

<210> 5

<211> 30

<212> DNA

< 213>artificial sequence

 

<220>

< 223>downstream primer of the R22-6 primer of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

 

<400> 5

tctagacaca?acgagagggc?tagatgaaag 30

 

<210> 6

<211> 36

<212> DNA

< 213>artificial sequence

 

<220>

< 223>upstream primer of the R22-7 primer of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

 

<400> 6

aagcttctag?ctcgagggcg?agcgtctaca?acaacc 36

 

<210> 7

<211> 30

<212> DNA

< 213>artificial sequence

 

<220>

< 223>downstream primer of the R22-7 primer of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

 

<400> 7

tctagacaca?acgagagggc?tagatgaaag 30

 

<210> 8

<211> 312

<212> DNA

< 213>corn (Zea mays L.)

 

<400> 8

cacaacgaga?gggctagatg?aaagcagttg?ttgttgttgt?acgtaggcgg?cagggttcac 60

catggctagt?tgactgagcg?ttggcagaaa?ctgttgtagc?tgttgttgtt?gctggtttgc 120

aactacgttc?gccagagcaa?gtgggttgga?tgcaagcagc?tgctcttgca?agtaggcggc 180

agggttcacc?atggctaggt?ggctcagtgc?tggtaggaac?tgttgttgct?gttgcgttgt 240

gatggcttgt?attgttagat?gtggcaagga?ttgttgttgc?aattgggcaa?ttggttgttg 300

tagacgctcg?cc 312

 

<210> 9

<211> 32

<212> DNA

< 213>artificial sequence

 

<220>

< 223>upstream primer of terminator poly (A)

 

<400> 9

ctcgagcgag?tttctccata?ataatgtgtg?ag 32

 

<210> 10

<211> 27

<212> DNA

< 213>artificial sequence

 

<220>

< 223>terminator poly (A) downstream primer

 

<400> 10

aagctttcgg?gggatctgga?ttttagt 27

 

<210> 11

<211> 185

<212> DNA

<213> Plasmid?p3301

 

<400> 11

tcgggggatc?tggattttag?tactggattt?tggttttagg?aattagaaat?tttattgata 60

gaagtatttt?acaaatacaa?atacatacta?agggtttctt?atatgctcaa?cacatgagcg 120

aaaccctata?ggaaccctaa?ttcccttatc?tgggaactac?tcacacatta?ttatggagaa 180

actcg 185

 

<210> 12

<211> 24

<212> DNA

< 213>artificial sequence

 

<220>

< 223>upstream primer of gus gene intron

 

<400> 12

tctagagttg?cctccctgct?gcgg 24

 

<210> 13

<211> 26

<212> DNA

< 213>artificial sequence

 

<220>

< 223>downstream primer of gus gene intron

 

<400> 13

tctagacggt?cagtggcagt?gaaggg 26

 

<210> 14

<211> 1023

<212> DNA

<213> Plasmid?pGreen-0229?Backbone

 

<400> 14

cattgttgcc?tccctgctgc?ggtttttcac?cgaagttcat?gccagtccag?cgtttttgca 60

gcagaaaagc?cgccgacttc?ggtttgcggt?cgcgagtgaa?gatccctttc?ttgttaccgc 120

caacgcgcaa?tatgccttgc?gaggtcgcaa?aatcggcgaa?attccatacc?tgttcaccga 180

cgacggcgct?gacgcgatca?aagacgcggt?gatacatatc?cagccatgca?cactgatact 240

cttcactcca?catgtcggtg?tacattgagt?gcagcccggc?taacgtatcc?acgccgtatt 300

cggtgatgat?aatcggctga?tgcagtttct?cctgccaggc?cagaagttct?ttttccagta 360

ccttctctgc?cgtttccaaa?tcgccgcttt?ggacatacca?tccgtaataa?cggttcaggc 420

acagcacatc?aaagagatcg?ctgatggtat?cggtgtgagc?gtcgcagaac?attacattga 480

cgcaggtgat?cggacgcgtc?gggtcgagtt?tacgcgttgc?ttccgccagt?ggcgcgaaat 540

attcccgtgc?accttgcgga?cgggtatccg?gttcgttggc?aatactccac?atcaccacgc 600

ttgggtggtt?tttgtcacgc?gctatcagct?ctttaatcgc?ctgtaagtgc?gcttgctgag 660

tttccccgtt?gactgcctct?tcgctgtaca?gttctttcgg?cttgttgccc?gcttcgaaac 720

caatgcctaa?agagaggtta?aagccgacag?cagcagtttc?atcaatcacc?acgatgccat 780

gttcatctgc?ccagtcgagc?atctcttcag?cgtaagggta?atgcgaggta?cggtaggagt 840

tggccccaat?ccagtccatt?aatgcgtggt?cgtgcaccat?cagcacgtta?tcgaatcctt 900

tgccacgcaa?gtccgcatct?tcatgacgac?caaagccagt?aaagtagaac?ggtttgtggt 960

taatcaggaa?ctgttcgccc?ttcactgcca?ctgaccggat?gccgacgcga?agcgggtaga 1020

tat 1023

 

<210> 15

<211> 5870

<212> DNA

< 213>artificial sequence

 

<220>

< 223>zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD

 

<400> 15

tcgcgcgttt?cggtgatgac?ggtgaaaacc?tctgacacat?gcagctcccg?gagacggtca 60

cagcttgtct?gtaagcggat?gccgggagca?gacaagcccg?tcagggcgcg?tcagcgggtg 120

ttggcgggtg?tcggggctgg?cttaactatg?cggcatcaga?gcagattgta?ctgagagtgc 180

accatatgcg?gtgtgaaata?ccgcacagat?gcgtaaggag?aaaataccgc?atcaggcgcc 240

attcgccatt?caggctgcgc?aactgttggg?aagggcgatc?ggtgcgggcc?tcttcgctat 300

tacgccagct?ggcgaaaggg?ggatgtgctg?caaggcgatt?aagttgggta?acgccagggt 360

tttcccagtc?acgacgttgt?aaaacgacgg?ccagtgaatt?cgagctcggc?aaagggactg 420

gcaaaggggc?ccactagagg?gttctttgcc?gagtgccagt?tcaccagaca?ctcggcaaat 480

aaacctcctt?tgccgagtgt?cgccgtgggc?actcggcgaa?ggatccgtca?ccgtcacttg 540

gcgccgtgac?ggtgactttt?ctttgccgag?tgccaaatgg?cactcggcaa?agtctttgcc 600

gagtgcccga?caaaaagtac?tcggcaaaga?tgctgttgcc?gatgtacagt?tcgccgagcg 660

ttctttgccg?agtgttacac?tcggcaaagc?atttgccgag?tgtaaaatag?cctttgccgt 720

gtgtctcaga?cacacggcaa?agaagctgat?tccggtagtg?tttggggggc?gactaacaca 780

aatcgatgac?ctttgatgaa?gatggatgta?tttctcccac?aaaatcttgg?atccatcctc 840

tactatgaaa?agtcatgaat?tcacaagacg?ttttcgaaaa?caatttcagc?ttcacttgta 900

aagttgcttg?tgcggctgaa?gccaagaaaa?cattagagga?caaacaataa?cttactatgc 960

ttttcgtgaa?ctgtttcaga?ccagcagtaa?taattattgg?tcctacatgc?attaatggat 1020

aagcggctag?catgccaaga?cggagcatta?gttaggagat?aagacctagc?gtttcattgc 1080

atccccatga?aaggaatact?atcactactg?catgaataat?tttcaaatgt?attaaaaact 1140

gatattgaaa?tgtaggtaat?ctgtccgcct?atgtgaaatg?tttaacagag?gcagccactc 1200

taggatgttg?cctctattaa?ttactcatag?atgcagccat?ccaactctag?gatttcaatt 1260

agtctcaatc?tatgcattcg?tctatgcatt?ctaggattac?aattagtctc?aatcttgtag 1320

tatttgttcc?ttccttgtca?aatcacttct?catctaacta?ctatgcttgt?ttaaccatca 1380

gaacataact?acaacaacat?ccatttataa?aggcttcgat?agcaaacttt?acatattcat 1440

atcatgttaa?ggttgtcaca?tgtgtaaagg?tgaagagatc?atgcatgtca?ttccacgtag 1500

ataaaaagaa?tgcctatata?aaaatggcac?attttcttgt?aggtagtgga?aagtatcttt 1560

ccagcaaaga?ccatataatc?cgataaagct?gataactaaa?tgtcgaaatc?gagtagatgc 1620

catatcatct?ataccttatc?tgttgtttgg?aaaaaaaaaa?gacaaaatcc?aaaaaaaaat 1680

ataagagatc?tcacctatat?aaatagctct?gaaatcagta?gttaatccat?cacccataat 1740

attttgagca?ttcagaaaca?caccaagcga?agcgcccggg?ggcgagcgtc?tacaacaacc 1800

aattgcccaa?ttgcaacaac?aatccttgcc?acatctaaca?atacaagcca?tcacaacgca 1860

acagcaacaa?cagttcctac?cagcactgag?ccacctagcc?atggtgaacc?ctgccgccta 1920

cttgcaagag?cagctgcttg?catccaaccc?acttgctctg?gcgaacgtag?ttgcaaacca 1980

gcaacaacaa?cagctacaac?agtttctgcc?aacgctcagt?caactagcca?tggtgaaccc 2040

tgccgcctac?gtacaacaac?aacaactgct?ttcatctagc?cctctcgttg?tgtctagaat 2100

atctacccgc?ttcgcgtcgg?catccggtca?gtggcagtga?agggcgaaca?gttcctgatt 2160

aaccacaaac?cgttctactt?tactggcttt?ggtcgtcatg?aagatgcgga?cttgcgtggc 2220

aaaggattcg?ataacgtgct?gatggtgcac?gaccacgcat?taatggactg?gattggggcc 2280

aactcctacc?gtacctcgca?ttacccttac?gctgaagaga?tgctcgactg?ggcagatgaa 2340

catggcatcg?tggtgattga?tgaaactgct?gctgtcggct?ttaacctctc?tttaggcatt 2400

ggtttcgaag?cgggcaacaa?gccgaaagaa?ctgtacagcg?aagaggcagt?caacggggaa 2460

actcagcaag?cgcacttaca?ggcgattaaa?gagctgatag?cgcgtgacaa?aaaccaccca 2520

agcgtggtga?tgtggagtat?tgccaacgaa?ccggataccc?gtccgcaagg?tgcacgggaa 2580

tatttcgcgc?cactggcgga?agcaacgcgt?aaactcgacc?cgacgcgtcc?gatcacctgc 2640

gtcaatgtaa?tgttctgcga?cgctcacacc?gataccatca?gcgatctctt?tgatgtgctg 2700

tgcctgaacc?gttattacgg?atggtatgtc?caaagcggcg?atttggaaac?ggcagagaag 2760

gtactggaaa?aagaacttct?ggcctggcag?gagaaactgc?atcagccgat?tatcatcacc 2820

gaatacggcg?tggatacgtt?agccgggctg?cactcaatgt?acaccgacat?gtggagtgaa 2880

gagtatcagt?gtgcatggct?ggatatgtat?caccgcgtct?ttgatcgcgt?cagcgccgtc 2940

gtcggtgaac?aggtatggaa?tttcgccgat?tttgcgacct?cgcaaggcat?attgcgcgtt 3000

ggcggtaaca?agaaagggat?cttcactcgc?gaccgcaaac?cgaagtcggc?ggcttttctg 3060

ctgcaaaaac?gctggactgg?catgaacttc?ggtgaaaaac?cgcagcaggg?aggcaacaat 3120

gtctagacac?aacgagaggg?ctagatgaaa?gcagttgttg?ttgttgtacg?taggcggcag 3180

ggttcaccat?ggctagttga?ctgagcgttg?gcagaaactg?ttgtagctgt?tgttgttgct 3240

ggtttgcaac?tacgttcgcc?agagcaagtg?ggttggatgc?aagcagctgc?tcttgcaagt 3300

aggcggcagg?gttcaccatg?gctaggtggc?tcagtgctgg?taggaactgt?tgttgctgtt 3360

gcgttgtgat?ggcttgtatt?gttagatgtg?gcaaggattg?ttgttgcaat?tgggcaattg 3420

gttgttgtag?acgctcgccc?tcgagcgagt?ttctccataa?taatgtgtga?gtagttccca 3480

gataagggaa?ttagggttcc?tatagggttt?cgctcatgtg?ttgagcatat?aagaaaccct 3540

tagtatgtat?ttgtatttgt?aaaatacttc?tatcaataaa?atttctaatt?cctaaaacca 3600

aaatccagta?ctaaaatcca?gatcccccga?aagcttggcg?taatcatggt?catagctgtt 3660

tcctgtgtga?aattgttatc?cgctcacaat?tccacacaac?atacgagccg?gaagcataaa 3720

gtgtaaagcc?tggggtgcct?aatgagtgag?ctaactcaca?ttaattgcgt?tgcgctcact 3780

gcccgctttc?cagtcgggaa?acctgtcgtg?ccagctgcat?taatgaatcg?gccaacgcgc 3840

ggggagaggc?ggtttgcgta?ttgggcgctc?ttccgcttcc?tcgctcactg?actcgctgcg 3900

ctcggtcgtt?cggctgcggc?gagcggtatc?agctcactca?aaggcggtaa?tacggttatc 3960

cacagaatca?ggggataacg?caggaaagaa?catgtgagca?aaaggccagc?aaaaggccag 4020

gaaccgtaaa?aaggccgcgt?tgctggcgtt?tttccatagg?ctccgccccc?ctgacgagca 4080

tcacaaaaat?cgacgctcaa?gtcagaggtg?gcgaaacccg?acaggactat?aaagatacca 4140

ggcgtttccc?cctggaagct?ccctcgtgcg?ctctcctgtt?ccgaccctgc?cgcttaccgg 4200

atacctgtcc?gcctttctcc?cttcgggaag?cgtggcgctt?tctcaatgct?cacgctgtag 4260

gtatctcagt?tcggtgtagg?tcgttcgctc?caagctgggc?tgtgtgcacg?aaccccccgt 4320

tcagcccgac?cgctgcgcct?tatccggtaa?ctatcgtctt?gagtccaacc?cggtaagaca 4380

cgacttatcg?ccactggcag?cagccactgg?taacaggatt?agcagagcga?ggtatgtagg 4440

cggtgctaca?gagttcttga?agtggtggcc?taactacggc?tacactagaa?gaacagtatt 4500

tggtatctgc?gctctgctga?agccagttac?cttcggaaaa?agagttggta?gctcttgatc 4560

cggcaaacaa?accaccgctg?gtagcggtgg?tttttttgtt?tgcaagcagc?agattacgcg 4620

cagaaaaaaa?ggatctcaag?aagatccttt?gatcttttct?acggggtctg?acgctcagtg 4680

gaacgaaaac?tcacgttaag?ggattttggt?catgagatta?tcaaaaagga?tcttcaccta 4740

gatcctttta?aattaaaaat?gaagttttaa?atcaatctaa?agtatatatg?agtaaacttg 4800

gtctgacagt?taccaatgct?taatcagtga?ggcacctatc?tcagcgatct?gtctatttcg 4860

ttcatccata?gttgcctgac?tccccgtcgt?gtagataact?acgatacggg?agggcttacc 4920

atctggcccc?agtgctgcaa?tgataccgcg?agacccacgc?tcaccggctc?cagatttatc 4980

agcaataaac?cagccagccg?gaagggccga?gcgcagaagt?ggtcctgcaa?ctttatccgc 5040

ctccatccag?tctattaatt?gttgccggga?agctagagta?agtagttcgc?cagttaatag 5100

tttgcgcaac?gttgttgcca?ttgctacagg?catcgtggtg?tcacgctcgt?cgtttggtat 5160

ggcttcattc?agctccggtt?cccaacgatc?aaggcgagtt?acatgatccc?ccatgttgtg 5220

caaaaaagcg?gttagctcct?tcggtcctcc?gatcgttgtc?agaagtaagt?tggccgcagt 5280

gttatcactc?atggttatgg?cagcactgca?taattctctt?actgtcatgc?catccgtaag 5340

atgcttttct?gtgactggtg?agtactcaac?caagtcattc?tgagaatagt?gtatgcggcg 5400

accgagttgc?tcttgcccgg?cgtcaatacg?ggataatacc?gcgccacata?gcagaacttt 5460

aaaagtgctc?atcattggaa?aacgttcttc?ggggcgaaaa?ctctcaagga?tcttaccgct 5520

gttgagatcc?agttcgatgt?aacccactcg?tgcacccaac?tgatcttcag?catcttttac 5580

tttcaccagc?gtttctgggt?gagcaaaaac?aggaaggcaa?aatgccgcaa?aaaagggaat 5640

aagggcgaca?cggaaatgtt?gaatactcat?actcttcctt?tttcaatatt?attgaagcat 5700

ttatcagggt?tattgtctca?tgagcggata?catatttgaa?tgtatttaga?aaaataaaca 5760

aataggggtt?ccgcgcacat?ttccccgaaa?agtgccacct?gacgtctaag?aaaccattat 5820

tatcatgaca?ttaacctata?aaaataggcg?tatcacgagg?ccctttcgtc 5870

 

<210> 16

<211> 29

<212> DNA

< 213>artificial sequence

 

<220>

< 223>upstream primer of the R19-4 primer of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

 

<400> 16

cccgggccat?ccaagcaagc?atcttacgg 29

 

<210> 17

<211> 26

<212> DNA

< 213>artificial sequence

<220>

< 223>downstream primer of the R19-4 primer of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

 

<400> 17

tctagaggca?taagggctcc?cgacaa 26

 

<210> 18

<211> 39

<212> DNA

< 213>artificial sequence

 

<220>

< 223>upstream primer of the R19-5 primer of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

 

<400> 18

aagcttctag?ctcgagccat?ccaagcaagc?atcttacgg 39

 

<210> 19

<211> 26

<212> DNA

< 213>artificial sequence

 

<220>

< 223>downstream primer of the R19-5 primer of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

 

<400> 19

tctagaggca?taagggctcc?cgacaa 26

 

<210> 20

<211> 286

<212> DNA

< 213>corn (Zea mays L.)

 

<400> 20

ccatccaagc?aagcatttac?ggtcattagc?attaaccctc?caacaaccat?atgctctatt 60

gcaacagcca?tccttagtgc?atctgtatct?ccaaagaatc?gcggcacaac?aactacaaca 120

acagttgcta?ccaacaatca?atcaagtagt?tgcagcgaac?cttgctgctt?acctccagca 180

acaacagttt?cttccattca?atcaactagc?tggggtgaac?cctgctatct?acttgcaggc 240

acaacagcta?ctaccattta?accaacttgt?cgggagccct?tatgcc 286

 

<210> 21

<211> 5819

<212> DNA

< 213>artificial sequence

 

<220>

< 223>zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD

 

<400> 21

tcgcgcgttt?cggtgatgac?ggtgaaaacc?tctgacacat?gcagctcccg?gagacggtca 60

cagcttgtct?gtaagcggat?gccgggagca?gacaagcccg?tcagggcgcg?tcagcgggtg 120

ttggcgggtg?tcggggctgg?cttaactatg?cggcatcaga?gcagattgta?ctgagagtgc 180

accatatgcg?gtgtgaaata?ccgcacagat?gcgtaaggag?aaaataccgc?atcaggcgcc 240

attcgccatt?caggctgcgc?aactgttggg?aagggcgatc?ggtgcgggcc?tcttcgctat 300

tacgccagct?ggcgaaaggg?ggatgtgctg?caaggcgatt?aagttgggta?acgccagggt 360

tttcccagtc?acgacgttgt?aaaacgacgg?ccagtgaatt?cgagctcggc?aaagggactg 420

gcaaaggggc?ccactagagg?gttctttgcc?gagtgccagt?tcaccagaca?ctcggcaaat 480

aaacctcctt?tgccgagtgt?cgccgtgggc?actcggcgaa?ggatccgtca?ccgtcacttg 540

gcgccgtgac?ggtgactttt?ctttgccgag?tgccaaatgg?cactcggcaa?agtctttgcc 600

gagtgcccga?caaaaagtac?tcggcaaaga?tgctgttgcc?gatgtacagt?tcgccgagcg 660

ttctttgccg?agtgttacac?tcggcaaagc?atttgccgag?tgtaaaatag?cctttgccgt 720

gtgtctcaga?cacacggcaa?agaagctgat?tccggtagtg?tttggggggc?gactaacaca 780

aatcgatgac?ctttgatgaa?gatggatgta?tttctcccac?aaaatcttgg?atccatcctc 840

tactatgaaa?agtcatgaat?tcacaagacg?ttttcgaaaa?caatttcagc?ttcacttgta 900

aagttgcttg?tgcggctgaa?gccaagaaaa?cattagagga?caaacaataa?cttactatgc 960

ttttcgtgaa?ctgtttcaga?ccagcagtaa?taattattgg?tcctacatgc?attaatggat 1020

aagcggctag?catgccaaga?cggagcatta?gttaggagat?aagacctagc?gtttcattgc 1080

atccccatga?aaggaatact?atcactactg?catgaataat?tttcaaatgt?attaaaaact 1140

gatattgaaa?tgtaggtaat?ctgtccgcct?atgtgaaatg?tttaacagag?gcagccactc 1200

taggatgttg?cctctattaa?ttactcatag?atgcagccat?ccaactctag?gatttcaatt 1260

agtctcaatc?tatgcattcg?tctatgcatt?ctaggattac?aattagtctc?aatcttgtag 1320

tatttgttcc?ttccttgtca?aatcacttct?catctaacta?ctatgcttgt?ttaaccatca 1380

gaacataact?acaacaacat?ccatttataa?aggcttcgat?agcaaacttt?acatattcat 1440

atcatgttaa?ggttgtcaca?tgtgtaaagg?tgaagagatc?atgcatgtca?ttccacgtag 1500

ataaaaagaa?tgcctatata?aaaatggcac?attttcttgt?aggtagtgga?aagtatcttt 1560

ccagcaaaga?ccatataatc?cgataaagct?gataactaaa?tgtcgaaatc?gagtagatgc 1620

catatcatct?ataccttatc?tgttgtttgg?aaaaaaaaaa?gacaaaatcc?aaaaaaaaat 1680

ataagagatc?tcacctatat?aaatagctct?gaaatcagta?gttaatccat?cacccataat 1740

attttgagca?ttcagaaaca?caccaagcga?agcgcccggg?ccatccaagc?aagcatttac 1800

ggtcattagc?attaaccctc?caacaaccat?atgctctatt?gcaacagcca?tccttagtgc 1860

atctgtatct?ccaaagaatc?gcggcacaac?aactacaaca?acagttgcta?ccaacaatca 1920

atcaagtagt?tgcagcgaac?cttgctgctt?acctccagca?acaacagttt?cttccattca 1980

atcaactagc?tggggtgaac?cctgctatct?acttgcaggc?acaacagcta?ctaccattta 2040

accaacttgt?cgggagccct?tatgcctcta?gaatatctac?ccgcttcgcg?tcggcatccg 2100

gtcagtggca?gtgaagggcg?aacagttcct?gattaaccac?aaaccgttct?actttactgg 2160

ctttggtcgt?catgaagatg?cggacttgcg?tggcaaagga?ttcgataacg?tgctgatggt 2220

gcacgaccac?gcattaatgg?actggattgg?ggccaactcc?taccgtacct?cgcattaccc 2280

ttacgctgaa?gagatgctcg?actgggcaga?tgaacatggc?atcgtggtga?ttgatgaaac 2340

tgctgctgtc?ggctttaacc?tctctttagg?cattggtttc?gaagcgggca?acaagccgaa 2400

agaactgtac?agcgaagagg?cagtcaacgg?ggaaactcag?caagcgcact?tacaggcgat 2460

taaagagctg?atagcgcgtg?acaaaaacca?cccaagcgtg?gtgatgtgga?gtattgccaa 2520

cgaaccggat?acccgtccgc?aaggtgcacg?ggaatatttc?gcgccactgg?cggaagcaac 2580

gcgtaaactc?gacccgacgc?gtccgatcac?ctgcgtcaat?gtaatgttct?gcgacgctca 2640

caccgatacc?atcagcgatc?tctttgatgt?gctgtgcctg?aaccgttatt?acggatggta 2700

tgtccaaagc?ggcgatttgg?aaacggcaga?gaaggtactg?gaaaaagaac?ttctggcctg 2760

gcaggagaaa?ctgcatcagc?cgattatcat?caccgaatac?ggcgtggata?cgttagccgg 2820

gctgcactca?atgtacaccg?acatgtggag?tgaagagtat?cagtgtgcat?ggctggatat 2880

gtatcaccgc?gtctttgatc?gcgtcagcgc?cgtcgtcggt?gaacaggtat?ggaatttcgc 2940

cgattttgcg?acctcgcaag?gcatattgcg?cgttggcggt?aacaagaaag?ggatcttcac 3000

tcgcgaccgc?aaaccgaagt?cggcggcttt?tctgctgcaa?aaacgctgga?ctggcatgaa 3060

cttcggtgaa?aaaccgcagc?agggaggcaa?caatgtctag?aggcataagg?gctcccgaca 3120

agttggttaa?atggtagtag?ctgttgtgcc?tgcaagtaga?tagcagggtt?caccccagct 3180

agttgattga?atggaagaaa?ctgttgttgc?tggaggtaag?cagcaaggtt?cgctgcaact 3240

acttgattga?ttgttggtag?caactgttgt?tgtagttgtt?gtgccgcgat?tctttggaga 3300

tacagatgca?ctaaggatgg?ctgttgcaat?agagcatatg?gttgttggag?ggttaatgct 3360

aatgaccgta?agatgcttgc?ttggatggct?cgagcgagtt?tctccataat?aatgtgtgag 3420

tagttcccag?ataagggaat?tagggttcct?atagggtttc?gctcatgtgt?tgagcatata 3480

agaaaccctt?agtatgtatt?tgtatttgta?aaatacttct?atcaataaaa?tttctaattc 3540

ctaaaaccaa?aatccagtac?taaaatccag?atcccccgaa?agcttggcgt?aatcatggtc 3600

atagctgttt?cctgtgtgaa?attgttatcc?gctcacaatt?ccacacaaca?tacgagccgg 3660

aagcataaag?tgtaaagcct?ggggtgccta?atgagtgagc?taactcacat?taattgcgtt 3720

gcgctcactg?cccgctttcc?agtcgggaaa?cctgtcgtgc?cagctgcatt?aatgaatcgg 3780

ccaacgcgcg?gggagaggcg?gtttgcgtat?tgggcgctct?tccgcttcct?cgctcactga 3840

ctcgctgcgc?tcggtcgttc?ggctgcggcg?agcggtatca?gctcactcaa?aggcggtaat 3900

acggttatcc?acagaatcag?gggataacgc?aggaaagaac?atgtgagcaa?aaggccagca 3960

aaaggccagg?aaccgtaaaa?aggccgcgtt?gctggcgttt?ttccataggc?tccgcccccc 4020

tgacgagcat?cacaaaaatc?gacgctcaag?tcagaggtgg?cgaaacccga?caggactata 4080

aagataccag?gcgtttcccc?ctggaagctc?cctcgtgcgc?tctcctgttc?cgaccctgcc 4140

gcttaccgga?tacctgtccg?cctttctccc?ttcgggaagc?gtggcgcttt?ctcaatgctc 4200

acgctgtagg?tatctcagtt?cggtgtaggt?cgttcgctcc?aagctgggct?gtgtgcacga 4260

accccccgtt?cagcccgacc?gctgcgcctt?atccggtaac?tatcgtcttg?agtccaaccc 4320

ggtaagacac?gacttatcgc?cactggcagc?agccactggt?aacaggatta?gcagagcgag 4380

gtatgtaggc?ggtgctacag?agttcttgaa?gtggtggcct?aactacggct?acactagaag 4440

aacagtattt?ggtatctgcg?ctctgctgaa?gccagttacc?ttcggaaaaa?gagttggtag 4500

ctcttgatcc?ggcaaacaaa?ccaccgctgg?tagcggtggt?ttttttgttt?gcaagcagca 4560

gattacgcgc?agaaaaaaag?gatctcaaga?agatcctttg?atcttttcta?cggggtctga 4620

cgctcagtgg?aacgaaaact?cacgttaagg?gattttggtc?atgagattat?caaaaaggat 4680

cttcacctag?atccttttaa?attaaaaatg?aagttttaaa?tcaatctaaa?gtatatatga 4740

gtaaacttgg?tctgacagtt?accaatgctt?aatcagtgag?gcacctatct?cagcgatctg 4800

tctatttcgt?tcatccatag?ttgcctgact?ccccgtcgtg?tagataacta?cgatacggga 4860

gggcttacca?tctggcccca?gtgctgcaat?gataccgcga?gacccacgct?caccggctcc 4920

agatttatca?gcaataaacc?agccagccgg?aagggccgag?cgcagaagtg?gtcctgcaac 4980

tttatccgcc?tccatccagt?ctattaattg?ttgccgggaa?gctagagtaa?gtagttcgcc 5040

agttaatagt?ttgcgcaacg?ttgttgccat?tgctacaggc?atcgtggtgt?cacgctcgtc 5100

gtttggtatg?gcttcattca?gctccggttc?ccaacgatca?aggcgagtta?catgatcccc 5160

catgttgtgc?aaaaaagcgg?ttagctcctt?cggtcctccg?atcgttgtca?gaagtaagtt 5220

ggccgcagtg?ttatcactca?tggttatggc?agcactgcat?aattctctta?ctgtcatgcc 5280

atccgtaaga?tgcttttctg?tgactggtga?gtactcaacc?aagtcattct?gagaatagtg 5340

tatgcggcga?ccgagttgct?cttgcccggc?gtcaatacgg?gataataccg?cgccacatag 5400

cagaacttta?aaagtgctca?tcattggaaa?acgttcttcg?gggcgaaaac?tctcaaggat 5460

cttaccgctg?ttgagatcca?gttcgatgta?acccactcgt?gcacccaact?gatcttcagc 5520

atcttttact?ttcaccagcg?tttctgggtg?agcaaaaaca?ggaaggcaaa?atgccgcaaa 5580

aaagggaata?agggcgacac?ggaaatgttg?aatactcata?ctcttccttt?ttcaatatta 5640

ttgaagcatt?tatcagggtt?attgtctcat?gagcggatac?atatttgaat?gtatttagaa 5700

aaataaacaa?ataggggttc?cgcgcacatt?tccccgaaaa?gtgccacctg?acgtctaaga 5760

aaccattatt?atcatgacat?taacctataa?aaataggcgt?atcacgaggc?cctttcgtc 5819

 

<210> 22

<211> 19

<212> DNA

< 213>artificial sequence

 

<220>

< 223>RNAi carrier pUC19-RNAi-22KD transforms T0 detects the 22Y3 primer for the PCR of regeneration plant upstream primer

      

<400> 22

caagccatca?caacgcaac 19

 

<210> 23

<211> 19

<212> DNA

< 213>artificial sequence

 

<220>

< 223>RNAi carrier pUC19-RNAi-22KD transforms T0 detects the 22Y3 primer for the PCR of regeneration plant downstream primer

      

<400> 23

cgtatccacg?ccgtattcg 19

 

<210> 24

<211> 19

<212> DNA

< 213>artificial sequence

 

<220>

< 223>RNAi carrier pUC19-RNAi-19KD transforms T0 detects the 19Y5 primer for the PCR of regeneration plant upstream primer

 

<400> 24

cagcgaagag?gcagtcaac 19

 

<210> 25

<211> 19

<212> DNA

< 213>artificial sequence

 

<220>

< 223>RNAi carrier pUC19-RNAi-19KD transforms T0 detects the 19Y5 primer for the PCR of regeneration plant downstream primer

      

<400> 25

cgaaccttgc?tgcttacct 19

 

 

Claims (2)

1. the construction process of a zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD may further comprise the steps:

(1) makes up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6

1. the acquisition of endosperm specificity expression promoter P-zp22/6

Genomic dna with corn variety is a template, is that primer is right with sequence shown in SEQ ID NO:1 and the SEQ ID NO:2, carries out pcr amplification, obtains the endosperm specificity expression promoter P-zp22/6 of sequence shown in SEQ ID NO:3;

2. make up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6 sequence

Cut the endosperm specificity expression promoter that has corresponding restriction enzyme site that step (1) 1. obtains with Sac I and Xba I enzyme; Be connected with the skeleton carrier pUC19 that cuts through Sac I and Xba I enzyme equally then, obtain having the recombinant vectors pUC19-p22/6 of endosperm specificity expression promoter P-zp22/6;

(2) make up the recombinant vectors pUC19-p22/6-22KD1 that inserts forward purpose fragment 22-KD-P and endosperm specificity expression promoter P-zp22/6

1. the acquisition of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

With the total RNA of corn kernel is template; With sequence shown in SEQ ID NO:4 and the SEQ ID NO:5; Carrying out the RT-PCR amplification, is template with this RT-PCR product then, is that primer is right with sequence shown in SEQ ID NO:6 and the SEQ ID NO:7; Carry out RT-PCR again, obtain the 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P shown in SEQ ID NO:8;

2. make up the recombinant vectors pUC19-p22/6-22KD1 that inserts forward purpose fragment 22-KD-P and endosperm specificity expression promoter P-zp22/6

1. the purpose fragment 22-KD-P that has corresponding restriction enzyme site that obtains with Sma I and Xba I double digestion step (2); Be connected with the same recombinant vectors pUC19-p22/6 that 2. makes up through the step (1) of Sma I and Xba I double digestion then, obtained inserting the recombinant vectors pUC19-p22/6-22KD1 of forward goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P and endosperm specificity expression promoter P-zp22/6;

(3) make up the recombinant vectors pUC19-22KD2 that inserts reverse purpose fragment 22-KD-P

Cut the purpose fragment 22-KD-P that has corresponding restriction enzyme site that step (2) 1. obtains with Hind III and Xba I enzyme; Be connected with the pUC19 carrier of cutting through Hind III and Xba I enzyme equally then, obtain inserting the recombinant vectors pUC19-22KD2 of reverse goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P;

(4) make up the recombinant vectors pUC19-22KD2-poly (A) that contains terminator poly (A)

With expression vector p3301 DNA is template, carries out pcr amplification with the terminator primer of sequence shown in SEQ ID NO:9 and the SEQ ID NO:10, obtains the terminator poly (A) of sequence shown in SEQ ID NO:11; The terminator poly (A) that further has corresponding restriction enzyme site with Xho I and Hind III double digestion; Be connected with the same recombinant vectors pUC19-22KD2 that obtains through the step (3) of Xho I and Hind III double digestion then, obtain containing the recombinant vectors pUC19-22KD2-poly (A) of terminator poly (A);

(5) structure contains endosperm specificity expression promoter P-zp22/6, the recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) of forward and reverse purpose fragment 22-KD-P and terminator poly (A)

The recombinant vectors pUC19-p22/6-22KD1 of step (2) structure and the recombinant vectors pUC19-22KD2-poly (A) of step (4) structure are used Xba I and Hind III double digestion respectively; The enzyme that will contain reverse purpose fragment 22-KD-P and terminator poly (A) is then cut product 22KD2-poly (A) and is connected on the recombinant vectors pUC19-p22/6-22KD1 that step (2) makes up; Obtain containing endosperm specificity expression promoter P-zp22/6, the recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) of forward and reverse goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P and terminator poly (A);

(6) structure of the connection of gus gene intron and zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD

With plasmid vector pGreen-0229 Backbone DNA is template, carries out pcr amplification with the gus gene intron primer of sequence shown in SEQ ID NO:12 and the SEQ ID NO:13, obtains the gus gene intron of sequence shown in SEQ ID NO:14; The gus gene intron that further has corresponding restriction enzyme site with Xba I single endonuclease digestion; Be connected with the recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) that phosphorylation processed steps (5) makes up behind Xba I single endonuclease digestion equally then; Obtain described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD, the nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD is shown in SEQ ID NO:15.

2. the construction process of a zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD may further comprise the steps:

(1) makes up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6

1. the acquisition of endosperm specificity expression promoter P-zp22/6

Genomic dna with corn variety is a template, is that primer is right with sequence shown in SEQ ID NO:1 and the SEQ ID NO:2, carries out pcr amplification, obtains the endosperm specificity expression promoter P-zp22/6 of sequence shown in SEQ ID NO:3;

2. make up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter zp22/6 sequence

Cut the endosperm specificity expression promoter that has corresponding restriction enzyme site that step (1) 1. obtains with Sac I and Xba I enzyme; Be connected with the skeleton carrier pUC19 that cuts through Sac I and Xba I enzyme equally then, obtain having the recombinant vectors pUC19-p22/6 of endosperm specificity expression promoter P-zp22/6;

(2) make up the recombinant vectors pUC19-p22/6-19KD1 that inserts forward purpose fragment 19-KD-P and endosperm specificity expression promoter P-zp22/6

1. the acquisition of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

With the total RNA of corn kernel is template; With the sequence shown in SEQ ID NO:16 and the SEQ ID NO:17; Carrying out the RT-PCR amplification, is template with this RT-PCR product then, is that primer is right with sequence shown in SEQ ID NO:18 and the SEQ ID NO:19; Carry out RT-PCR again, obtain the 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P of sequence shown in SEQ ID NO:20;

2. make up the recombinant vectors pUC19-p22/6-19KD1 that inserts forward purpose fragment 19-KD-P and endosperm specificity expression promoter P-zp22/6

1. the purpose fragment 19-KD-P that has corresponding restriction enzyme site that obtains with Sma I and Xba I double digestion step (2); Be connected with the same recombinant vectors pUC19-p22/6 that 2. makes up through the step (1) of Sma I and Xba I double digestion then, obtained inserting the recombinant vectors pUC19-p22/6-19KD1 of forward goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P and endosperm specificity expression promoter P-zp22/6;

(3) make up the recombinant vectors pUC19-19KD2 that inserts reverse purpose fragment 19-KD-P

Cut the purpose fragment 19-KD-P that has corresponding restriction enzyme site that step (2) 1. obtains with Hind III and Xba I enzyme; Be connected with the pUC19 carrier of cutting through Hind III and Xba I enzyme equally then, obtain inserting the recombinant vectors pUC19-19KD2 of reverse goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P;

(4) make up the recombinant vectors pUC19-19KD2-poly (A) that contains terminator poly (A)

With expression vector p3301 DNA is template, carries out pcr amplification with the terminator primer of sequence shown in SEQ ID NO:9 and the SEQ ID NO:10, obtains the terminator poly (A) of sequence shown in SEQ ID NO:11; The terminator poly (A) that further has corresponding restriction enzyme site with Xho I and Hind III double digestion; Be connected with the same recombinant vectors pUC19-19KD2 that obtains through the step (3) of Xho I and Hind III double digestion then, obtain containing the recombinant vectors pUC19-19KD2-poly (A) of terminator poly (A);

(5) structure contains endosperm specificity expression promoter P-zp22/6, the recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) of forward and reverse purpose fragment 19-KD-P and terminator poly (A)

The recombinant vectors pUC19-p22/6-22KD1 of step (2) structure and the recombinant vectors pUC19-19KD2-poly (A) of step (4) structure are used Xba I and Hind III double digestion respectively; The enzyme that will contain reverse purpose fragment 19-KD-P and terminator poly (A) is then cut product 19KD2-poly (A) and is connected on the recombinant vectors pUC19-p22/6-19KD1 that step (2) makes up; Obtain containing endosperm specificity expression promoter P-zp22/6, the recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) of forward and reverse goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P and terminator poly (A);

(6) structure of the connection of gus gene intron and zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD

With plasmid vector pGreen-0229 Backbone DNA is template, carries out pcr amplification with the gus gene intron primer of sequence shown in SEQ ID NO:12 and the SEQ ID NO:13, obtains the gus gene intron of sequence shown in SEQ ID NO:14; The gus gene intron that further has corresponding restriction enzyme site with Xba I single endonuclease digestion; Be connected with the recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) that phosphorylation processed steps (5) makes up behind Xba I single endonuclease digestion equally then; Obtain described zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD, the nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD is shown in SEQ ID NO:21.

Images