CN110129322B - Bna-miR393 application in regulation and control of brassica napus reproductive organ development process - Google Patents

Abstract

The invention relates to application of Bna-miR393 in regulation and control of a cabbage type rape reproductive organ development process. In particular relates to a DNA fragment containing Bna-miR393 precursor sequence (pre-miR 393) which has a sequence shown as SEQ ID NO. 1, a sequence length of 618bp and a corresponding mature miRNA sequence shown as SEQ ID NO. 2. Functional analysis of cabbage type rape genetic transformation shows that Bna-miR393 participates in regulation and control of development of cabbage type rape reproductive organs, and the method has very important significance for genetic breeding improvement of cabbage type rape.

Description

Bna-miR393 application in regulation and control of brassica napus reproductive organ development process

Technical Field

The invention relates to the field of cabbage type rape genetic engineering. In particular to the application of the cabbage type rape Bna-miR393 capable of influencing the development of reproductive organs, which is obtained by separation, cloning and functional verification, in the genetic breeding improvement of the cabbage type rape. According to the invention, a DNA fragment containing a cabbage type rape Bna-miR393 precursor sequence is separated by adopting an RT-PCR method, and overexpression of Bna-miR393 can influence the development of cabbage type rape reproductive organs, so that the function and application approach of the miRNA are proved.

Background

Cabbage type rape (Brassica napusL.) is a main rape cultivation variety in China, and has the characteristics of high yield, strong stress resistance, wide adaptability and the like. mirnas are a class of endogenous, non-coding small RNAs of about 21-24 nt in length. The plant miRNA is obtained by cutting and processing an initial transcript by DCL1 cutting enzyme, a mature miRNA sequence is combined with an RNA-mediated silencing complex, a target gene which is complementary with the miRNA sequence is identified, and finally the target gene is regulated and controlled by cutting or translating and inhibiting the target gene. With the development of high-throughput sequencing technology, a great deal of research finds that miRNA participates in multiple aspects of Plant growth and development, including seed germination, leaf morphology, flower differentiation and development, root development, transition from vegetative stage to reproductive stage, and the like (D' Ario M, Griffiths-Jones S, Kim M. Small RNAs: big impact on Plant development. Trends Plant Sci, 2017, 22(12): 1056-; in addition, miRNA also has important roles in plant disease resistance, abiotic stress resistance and the like. miR393 is a conserved miRNA family in plants, target genes of the miRNA family code auxin receptor proteins TIR1, AFB1/2/3 and the like, the proteins participate in auxin signal perception and Aux/IAA protein degradation, and the Aux/IAA protein can inhibit auxin related gene expression through interaction with ARF and the like, so that the plant growth and development and the response to biotic and abiotic stresses (Parry G, Calderon-Villalobos LI, Prigge M, et aleptors, Proc Natl Acad Sci USA, 2009, 106(52): 22540-. Research shows that miR393 plays a key role in the antibacterial property process of plants, and the over-expression of miR393 in Arabidopsis can improve the resistance of the plants to bacterial infection of exogenous pathogenic bacteria (Navarro L, Dunoyer P, Jay F, et al. A plant miRNA controls to antibacterial resistance by repressing auxiliary signalling Science, 2006, 312(5772): 436).

The cabbage type rape with completely different reproductive organ morphological phenotypes obtained by utilizing a transgenic technology is not reported at present.

Disclosure of Invention

The invention aims to provide the sequence and the function of the brassica napus Bna-miR393, and further discloses application of the miRNA in regulation and control of the development of the reproductive organs of the brassica napus.

According to the invention, the cabbage type rape is taken as a research material, and RNA-seq comparison analysis is carried out on different development periods of reproductive organs of the cabbage type rape at the early stage, so that Bna-miR393 is obviously and differentially expressed in the development process of the reproductive organs of the cabbage type rape, and the miRNA is shown to possibly participate in regulation and control of the development of the reproductive organs of the cabbage type rape. Therefore, the method has very important significance for the genetic breeding improvement of the brassica napus by separating a DNA fragment containing the precursor sequence of the brassica napus Bna-miR393 from the brassica napus and identifying the function of Bna-miR393 in the development process of the reproductive organs of the brassica napus.

The invention separates and applies a DNA segment containing Bna-miR393 precursor sequence (also called precursor miRNA, the precursor miRNA (pre-miRNA) is a part of 618 bp), the sequence is shown as SEQ ID NO:1, the sequence length is 618bp, the corresponding miRNA mature sequence is shown as SEQ ID NO:2, and the mature miRNA has the capability of regulating and controlling the development of cabbage type rape reproductive organs. The agrobacterium-mediated method is utilized to be transformed into wild cabbage type rape plants, and the biological function of the wild cabbage type rape plants in bodies of cabbage type rape plants is researched, so that a good foundation is provided for the application of Bna-miR393 in cabbage type rape genetic breeding.

The invention aims to obtain a mature miRNA for regulating and controlling the development of the reproductive organ of Brassica napus, wherein the sequence of the mature miRNA is shown as SEQ ID NO. 2.

It is a further object of the present invention to provide an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line comprising the aforementioned DNA fragment.

It is a further object of the present invention to provide the use of (a 1) or (a 2):

(a1) the application of the brassica napus Bna-miR393, or the DNA fragment, or the expression cassette, the recombinant vector, the recombinant microorganism or the transgenic cell line in regulation and control of the development of the reproductive organs and the morphological phenotype of the reproductive organs of the brassica napus;

(a2) the brassica napus Bna-miR393, or the DNA fragment, or the expression cassette, the recombinant vector, the recombinant microorganism or the transgenic cell line are applied to the brassica napus genetic breeding.

The invention also provides a method for cultivating the transgenic plant for changing the reproductive organ form of the target plant, which aims to improve the content of the miRNA in the target plant and obtain the transgenic plant; transforming a target plant by using the DNA fragment containing the precursor sequence of the Brassica napus Bna-miR393 to obtain a transgenic plant; the reproductive organ morphological phenotype of the transgenic plant is significantly different from that of the target plant.

The cultivation method specifically comprises the following steps:

(1) cloning a DNA fragment containing a precursor sequence of Brassica napus Bna-miR 393;

(2) transforming the plasmid with the cabbage type rape Bna-miR393 precursor sequence into agrobacterium by an electric shock method;

(3) and (3) transforming the agrobacterium with the transformation plasmid into a target plant by adopting a transgenic method to obtain a transgenic plant.

The expression vector carrying the DNA fragment containing the precursor sequence of Bna-miR393 of the present invention can be introduced into Plant cells by conventional biotechnological methods using Ti plasmids, Plant viral vectors, direct DNA transformation, microinjection, electroporation and the like (Weissbach, 1998, Method for Plant Molecular Biology VIII, academic Press, New York, pp.411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition)).

A host (various plants including Brassica napus) can be transformed by using the expression vector comprising the DNA fragment containing the Bna-miR393 precursor sequence, and plant varieties with improved plant types can be cultivated.

And (2) recovering the DNA fragment containing the Bna-miR393 precursor sequence by using a DNA recovery kit, connecting the fragment into a pMDC83 skeleton vector by using an enzyme digestion connection method, and constructing an over-expression vector of the DNA fragment, wherein the over-expression vector is named pMDC83-miR 393.

The pMDC83-miR393 vector is introduced into Agrobacterium tumefaciens by an electrotransformation method, and the Agrobacterium tumefaciens strain is GV 3101. Through an agrobacterium infection mediated genetic transformation method, pMDC83-miR393 is transformed into a cabbage type rape receptor material J9712, a transgenic plant with the expression quantity of Bna-miR393 remarkably improved relative to a wild type is successfully obtained, and observation shows that compared with the wild type plant, the morphology of a reproductive organ of the transgenic cabbage type rape with over-expression Bna-miR393 is obviously changed, which indicates that Bna-miR393 can regulate and control the development of the reproductive organ of the plant.

In conclusion, the invention uses the brassica napus as a research material, and discovers a candidate miRNA capable of regulating and controlling the development of the reproductive organs of the brassica napus by analyzing the phenotypic change of the reproductive organs of transgenic brassica napus plants over-expressing Bna-miR393, wherein the miRNA possibly plays an important role in the development process of the reproductive organs and provides an important theoretical basis for the genetic breeding of the brassica napus.

Drawings

SEQ ID NO 1 of the sequence table is a DNA fragment which is separated and cloned and contains Bna-miR393 precursor sequence, and the length of the DNA fragment is 618 bp;

the sequence table SEQ ID NO. 2 is a mature Bna-miR393 sequence;

FIG. 1 Bna-miR393 expression profile in various tissues of Brassica napus;

FIG. 2 expression of Bna-miR393 in 2 Bna-miR393 overexpressing plants, in which: WT is wild type control plant; l1, L2, L7 and L15 are Bna-miR393 transgenic cabbage type rape;

FIG. 3 Bna-miR393 overexpresses the reproductive organ phenotype of transgenic Brassica napus, in which: WT is wild type control plant; l1, L2, L7, L15 are Bna-miR393 transgenic brassica napus. A: the floral organ; b: carrying out silique; c: seeds; d: area of petals; e: (ii) silique length; f: seed diameter; g: thousand grains are heavy.

Detailed Description

The following example defines the invention and describes the method of the invention to clone Bna-miR393 precursor sequences and to verify Bna-miR393 function. From the following description and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Example 1: qRT-PCR analysis Bna-miR393 expression condition in various tissues and organs of Brassica napus

Taking different tissue samples of the cabbage type rape in each period, quickly placing the samples in liquid nitrogen for quick freezing, and transferring the samples to a refrigerator at 70 ℃ for storage until RNA is extracted. Total RNA was extracted using the RNAioso Plus kit from TaKaRa. The expression level of Bna-393 miR was subjected to qPCR Detection by poly (A) tailing method using miRNA reverse transcription Kit (miRcute Plus miRNA First-StrandcDNA Synthesis Kit) and expression level Detection Kit SYBR Green (miRcute Plus miRNA qPCR Detection Kit, including reverse universal primer). The upstream primer is a mature sequence of Bna-miR 393: 5'-TCCAAAGGGATCGCATTGATC-3' (SEQ ID NO: 3), and the downstream primer is provided by the kit. 5.8S RNA is used as an internal reference gene, and the used internal reference primers are as follows: 5'-GTCTGCCTGGGTGTCACG-3' (SEQ ID NO: 4), and qPCR analysis was performed using an ABI7500 fluorescent quantitative PCR instrument. The reaction procedure is as follows: collecting fluorescence signals at 95 ℃ for 10 min, 95 ℃ for 20 s and 58 ℃ for 34 s for 40 cycles; fluorescence signals were collected every 1 ℃ for 1s from 60 ℃ to 95 ℃. Each sample is provided with 3 technical repetitions, and after the reaction is finished, analysis and mapping are carried out by ABI7500 self-contained Software (7500 Software v2.0.1), and the relative expression quantity of Bna-miR393 in each tissue organ of the brassica napus is calculated, as shown in figure 1.

Example 2: molecular cloning of Brassica napus Bna-miR393 precursor sequence

Taking three-leaf one-heart-stage seedlings of the cabbage type rape variety Darmor-bzh, quickly freezing the seedlings by liquid nitrogen, and storing the seedlings in a refrigerator at the temperature of 70 ℃ below zero for extracting total RNA. Total RNA was extracted using the RNAioso Plus kit from TaKaRa, Inc. Synthesis of cDNA for Brassica napus first Strand Synthesis was performed according to HiScript 1st Strand cDNA Synthesis Kit, Inc., of Biotech, Inc., Nanjing Novodka.

Using the first strand of cDNA as an amplification template, designing F: 5'-AAGTTAAAGATGAGAAGG-3' (SEQ ID NO: 5) and R: 5'-TTCAAGATGGGTCAG ATTCT-3' (SEQ ID NO: 6) as primers, and performing cDNA amplification by RT-PCR under the following conditions: 3 min at 94 ℃, 15 s at 59 ℃ and 30 s at 72 ℃ for 35 cycles; 10 min at 72 ℃. And (3) carrying out electrophoretic analysis after the PCR is finished, and recovering the target amplified fragment by adopting a DNA recovery kit of Kangji biological technology limited company. Connecting the amplified fragment with pEASY-Blunt T vector of Beijing Quanji Biotechnology Limited, transforming competent cells of Escherichia coli, selecting white colony to perform colony PCR to identify positive clone, sending the positive clone to Yangzhou Ongke Biotechnology Limited for sequencing, and designating the plasmid without errors after sequencing verification as pre-393 miR-T.

Example 3: Bna-miR393 overexpression vector construction

To better analyze the function of Bna-miR393, applicants over-expressed it in Brassica napus and studied the function of this miRNA by observing the phenotype of the transgenic plants.

The construction method of the overexpression vector comprises the following steps: taking the Bna-miR393 precursor sequence cloning vector plasmid pre-miR393-T which is verified to be error-free by sequencing as a template, and using a primer pre-miR 393F: 5'-TTAATTAAAAGTTAAAGATGAGAAGG-3') (SEQ ID NO: 7) (sequence specific primer plus linkerPac Site I) and pre-miR 393R: 5'-GGCGCGCCTTCAGATGGTCAGAT TCT-3') (SEQ ID NO: 8) (sequence specific primer plus linkerAsc I site), and carrying out DNA amplification by using PCR under the following conditions: 3 min at 94 ℃, 15 s at 60 ℃ and 30 s at 72 ℃ for 35 cycles; 10 min at 72 ℃. Performing electrophoresis analysis after PCR is finished, and adopting Kangji century biotechnology limited methodThe DNA recovery kit of department retrieves the amplified fragment of interest. Connecting the amplified fragment to pEASY-Blunt T vector of Beijing holotype gold biotechnology, Inc., transforming escherichia coli competent cells, selecting white colony to perform colony PCR to identify positive clone, and sending the positive clone to Yangzhou Ongke Biotechnology, Inc. for sequencing. Cloning vector plasmid containing Bna-miR393 precursor sequencePac I、Asc After the double enzyme digestion, a DNA recovery kit is used for recovering a target DNA fragment, and the fragment is connected with a pMDC83 skeleton vector which is correspondingly subjected to enzyme digestion to construct an over-expression vector of Bna-miR393, which is named as pMDC83-miR 393.

Example 4: genetic transformation of brassica napus with pMDC83-miR393 overexpression vector

The pMDC83-miR393 plasmid is introduced into competent cells of Agrobacterium tumefaciens GV3101 strain by electric shock transformation. Selecting single colony, inoculating into 25 mL YEB culture medium (containing 50 mg/L rifampicin), culturing overnight, inoculating 5 mL bacterial liquid into 100 mL YEB culture medium (containing 50 mg/L rifampicin), and culturing to OD ₆₀₀ And (5) putting the bacterial liquid on ice for 10 min, centrifuging at 5000 rpm and 4 ℃ for 10 min to collect thalli, and adding 100 mL sterile double distilled water to wash twice. The cells were suspended in 4 mL of 10% glycerol and transferred to a 50 mL centrifuge tube. The thalli is collected by centrifugation at 5500 rpm at 4 ℃ for 10 min, 500 muL of 10% glycerol is added to resuspend the thalli, and the thalli is transferred to a 1.5 mL centrifuge tube.

And taking 50 muL of competent cells, adding 5 muL of pMDC83-miR393 recombinant plasmid, uniformly mixing by using a gun head, and transferring to a 0.1 cm electric transformation cup. Electrical conversion parameters: 200 omega, 1.7 KV, 2.5F, and 500 muL of LB culture solution is added immediately after electric shock. After culturing at 37 ℃ and 220 rpm for 1 h, 100 muL of bacterial liquid is taken and coated on LB culture medium containing Kanamycin Kanamycin resistance to screen transformants, and culturing is carried out for 16 h at 28 ℃.

The genetic transformation method of the cabbage type rape is a method for referencing improvement of a transformation method of a key laboratory of the national crop genetic improvement of Huazhong agriculture university, the hypocotyl of the sterile seedling of the cabbage type rape is used as an explant, and the genetic transformation of an exogenous fragment in the cabbage type rape is realized by utilizing an agrobacterium-mediated method.

The formula of the culture medium is as follows:

inoculation Medium (M) ₀ ）：MURASHIGE &SKOOG MEDIUM (Duchefa Biochemie company) +30.0 g/L Sucrose +8 g/L Agar (pH 5.8-pH 6.0).

Co-cultivation Medium (M) ₁ ）：M ₀ + 18.0 g/L Mannitol Mannitol + 1.0 mg/L2, 4-dichlorophenoxyacetic acid 2,4-D +0.3 mg/L Kinetin Kinetin + 100. mu.M acetosyringone AS (pH 5.8).

Callus differentiation medium (M) ₂ ）：M ₁ +300.0 mg/L Timentin +25 mg/L Hygromycin B.

Seedling culture medium (M) ₃ ）：MURASHIGE &SKOOG MEDIUM (Duchefa Biochemie company) +10.0 g/L Glucose +0.25 g/L Xylose xylosyl +0.6 g/L morpholine ethanesulfonic acid MES +2.0 mg/L Zeatin +0.1 mg/L indoleacetic acid IAA +300.0 mg/L Timentin +25 mg/L Hygromycin B.

Strong seedling rooting culture medium (M) ₄ ）：M ₀ +300.0 mg/L Timentin.

Murashige & Skoog media is simply referred to as MS MEDIUM.

The specific operation steps are as follows:

(1) and (3) sterilization:

a. firstly, soaking cabbage type rape seeds in 75% alcohol for 1 min, wherein attention cannot be paid for too long time;

b. then sterilizing with 2% sodium hypochlorite for 20 min;

c. finally, the seeds are washed for 4-5 times by sterile water, and are cleaned as far as possible.

(2) Sowing:

a. seeding the sterilized seeds to M with sterile forceps ₀ 30 grains per dish on the culture medium;

b. placing the inoculated culture tank into an incubator, and performing dark culture at 24 ℃ for 6-7 d.

(3) Shaking the bacteria:

after 5-6 days of sowing, inoculating the agrobacterium into a sterile triangular flask or a centrifuge tube containing LB liquid culture medium, and placing the sterile triangular flask or the centrifuge tube into a shaker at 28 ℃ and 180 and 220 rpm for culturing.

(4) Explants were prepared and infected:

a. cutting the seedling which grows for 6-7 d after seeding by using sterile forceps and a scalpel, and cutting the hypocotyl into explant segments with the length of 0.8-1.0 cm. When cutting seedling, the hypocotyl is arranged in M ₁ The liquid culture medium is cut, so that the cutting effect is better. The cutting is fast and accurate without dragging;

b. measuring OD of Agrobacterium ₆₀₀ Value (OD in LB medium) ₆₀₀ = about 0.3 is preferable), the previously cultured bacterial solution is centrifuged at 6000 rpm for 10 min, the supernatant is discarded, the bacterial solution is resuspended in an MS liquid medium containing 100. mu.M acetosyringone AS, which is the same volume AS the bacterial solution, and the process is repeated once more. Finally, taking 2 mL of bacterial liquid, and diluting the bacterial liquid with 20 mL of MS liquid culture medium containing 100 mu M acetosyringone AS;

c. and (3) putting the cut explants into the bacterial liquid resuspension with the adjusted concentration, and carrying out dip dyeing for 10 min, wherein the infection time is not too long, otherwise, the explants die. 150-200 explants are suitably impregnated in each 20 mL of the bacterial solution;

(5) transfer of infected explants to M ₁ Culturing on culture medium at 24 deg.C in dark for 36-48 hr with 20-25 explants per dish;

(6) explants were transferred from M1 to M2 medium and transferred to a light incubator (24 ℃, 16 h/8 h light/dark) for 3 weeks;

(7) transfer of explants to M ₃ Subculturing on the culture medium every 2-3 weeks until green buds appear;

(8) finally transferring the explants to M ₄ Rooting in culture medium for 2-4 weeks.

Example 5: identification of transgenic cabbage type rape positive plants

The method for extracting the genome DNA of the brassica napus by adopting the method for quickly extracting the plant DNA comprises the following specific steps:

(1) taking two young leaves (about 0.2 g), shearing and putting into a2 mL centrifuge tube, adding 250 mu L of DNA buffer (500 mM Tris-HCl, 300 mM NaCl, 300 mM Cross, pH = 7.5) and two steel balls (diameter 6.7 mM), and breaking a leaf sample by a sample making machine for 50 Hz and 180 s;

(2) incubating the fragmented sample at 95 ℃ for 10 min;

(3) taking out the sample, cooling to room temperature, and centrifuging at 12000 rpm for 5 min;

(4) and (4) sucking 50 muL of supernatant, transferring the supernatant into a new 1.5 mL centrifuge tube, and diluting by 5 times for later use.

Taking 1 mu L DNA as a template, and carrying out PCR amplification by using primers 35S (5'-TCCCACTATCCTTCGCAAG-3') (SEQ ID NO: 9) and GFP (5'-tcagggtaacgggagaagc-3') (SEQ ID NO: 10), wherein the amplification conditions are as follows: 5 min at 94 ℃; 30 cycles of 94 ℃ for 30 s, 58 ℃ for 30 s and 72 ℃ for 30 s; 10 min at 72 ℃. By using the transgenic cabbage type rape DNA as a template, a specific target segment can be amplified, and the integration of a target vector pMDC83-miR393 into a cabbage type rape genome is proved.

Example 6: overexpression of Bna-miR393 enables reproductive organ morphology of transgenic brassica napus to be changed

The invention adopts a fluorescent real-time quantitative PCR method to detect the expression of Bna-miR393 in part of transgenic cabbage type rape plants, and the RNA extraction and Bna-miR393 expression amount detection method are shown in example 1. The result shows that the transgenic plant with the expression level of Bna-miR393 remarkably improved relative to that of the wild-type Bna-miR393 is successfully obtained (figure 2). Bna-miR393 expression level of miR393 in the over-expression plant is improved by 3-9 times compared with that of a control.

Phenotype analysis is carried out on the transgenic plant over-expressing Bna-miR393, and the result shows that compared with a wild control plant, the morphology of the reproductive organ of the transgenic plant over-expressing Bna-miR393 is obviously changed, namely the floral organ is reduced, the silique is reduced, and the seeds are reduced (figure 3).

Bna-miR393 overexpression plants have smaller flower organs than a control, and the petal areas are 21% -54% smaller than the control; the length of the horn is reduced by 25% -46% compared with the control; the grain size is reduced by 4% -12% compared with the control; the thousand grain weight is reduced by 6-26% compared with the control.

The invention separates and obtains the Brassica napus miRNA related to the development of reproductive organs, can pertinently obtain the candidate miRNA for regulating and controlling the development of the plant reproductive organs, and has certain theoretical guidance function for researching the crop yield and separating the miRNA related to the development of the plant reproductive organs. The miRNA related to reproductive organ development obtained by separation is from the plant, and has small influence on the environment. The function research of the separated miRNA is utilized, a basis can be provided for the breeding improvement of the rape molecules, and the method has very important significance for cultivating a new cabbage type rape variety and changing the yield and the character of the cabbage type rape.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

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Claims (4)

1. The application of the DNA fragment of the precursor sequence of the brassica napus Bna-miR393 in regulation and control of the morphological phenotype of the reproductive organ of the brassica napus is characterized in that the sequence of the DNA fragment is shown as SEQ ID NO. 1, and the mature sequence of the brassica napus Bna-miR393 is shown as SEQ ID NO. 2; the reproductive organ morphological phenotype includes floral organ size, silique length, and seed size.

2. The use according to claim 1, wherein a DNA fragment of a precursor sequence of brassica napus Bna-miR393 according to claim 1 is used to transform a target plant to obtain a transgenic plant; the target plant is cabbage type rape; the reproductive organ morphology and phenotype of the transgenic plant are obviously different from those of the target plant.

3. Use according to claim 1, characterized in that it comprises the following steps: (1) cloning a DNA fragment containing a precursor sequence of Brassica napus Bna-miR 393;

(2) transforming the plasmid with the cabbage type rape Bna-miR393 precursor sequence into agrobacterium by an electric shock method;

(3) and (3) transforming the agrobacterium with the transformation plasmid into a target plant by adopting a transgenic method to obtain a transgenic plant.

4. The use of claim 3, wherein the primers SEQ ID NO 5 and SEQ ID NO 6 are used for cDNA amplification by RT-PCR.

Images