阅读说明：本技术 植物转基因筛选载体pCALSm1及其应用 (Plant transgenic screening vector pCALSm1 and application thereof ) 是由 欧阳超 安保光 陈思兰 赵惠敏 李丹 龙湍 吴永忠 黄培劲 于 2020-05-07 设计创作，主要内容包括：本发明涉及农业生物技术领域,具体涉及植物转基因筛选载体pCALSm1及其应用。本发明提供的植物遗传转化筛选载体利用包含水稻乙酰乳酸合成酶突变基因的表达盒作为筛选标记,在愈伤组织筛选阶段使用嘧啶水杨酸类、咪唑啉酮类等除草剂作为筛选剂,所获得的转基因植株具备对嘧啶水杨酸类、咪唑啉酮类等除草剂的高抗性。该载体可作为转基因筛选载体附加其他功能元件使用,在转基因过程中利用植物内源基因,不引入菌源等外源筛选标记基因,不仅丰富了植物转基因筛选方法,还可有效降低外源基因引起的转基因植物的潜在安全风险以及公众对转基因植物的担忧,有利于转基因植物的商业化应用,市场价值和社会效益均良好。(The invention relates to the technical field of agricultural biology, in particular to a plant transgenic screening vector pCALSm1 and application thereof. The plant genetic transformation screening vector provided by the invention uses an expression cassette containing a rice acetolactate synthase mutant gene as a screening marker, and herbicides such as pyrimidinylsalicylic acid and imidazolinone are used as screening agents in a callus screening stage, so that the obtained transgenic plant has high resistance to the herbicides such as pyrimidinylsalicylic acid and imidazolinone. The vector can be used as a transgenic screening vector with other additional functional elements, plant endogenous genes are utilized in the transgenic process, exogenous screening marker genes such as bacterial sources and the like are not introduced, the transgenic screening method of the plant is enriched, potential safety risks of the transgenic plant caused by the exogenous genes and public concerns about the transgenic plant can be effectively reduced, the commercial application of the transgenic plant is facilitated, and the market value and the social benefit are good.)

1. A plant transgenic screening expression cassette is characterized in that a plant endogenous gene is used as a screening marker, and the plant endogenous gene is an ALS mutant gene.

2. The plant transgene screening expression cassette of claim 1, wherein the ALS mutant gene is a rice ALS mutant gene;

preferably, the nucleotide sequence of the rice ALS mutant gene is shown as SEQ ID NO. 1.

3. The plant transgene screening expression cassette of claim 1 or 2, further comprising a promoter and a terminator for initiating and terminating transcription of the ALS mutant gene;

the promoter is a rice ALS gene promoter, a rice or corn Ubi promoter, an Actin promoter, a Rubisco small subunit promoter or a Cab promoter;

the terminator is a rice ALS gene terminator or a Ubi terminator;

preferably, the promoter is a rice ALS gene promoter; the terminator is a rice Ubi terminator.

4. The plant transgene screening expression cassette of any one of claims 1 to 3, wherein the nucleotide sequence of the expression cassette is shown as SEQ ID No. 2.

5. A plant genetic transformation screening vector comprising the plant transgene screening expression cassette according to any one of claims 1 to 4.

6. The plant genetic transformation screening vector according to claim 5, which is a plant binary expression vector into which genetic transformation can be performed by cloning other expression cassettes;

the other expression cassette is an expression cassette other than the plant transgene screening expression cassette according to any one of claims 1 to 4.

7. The plant genetic transformation screening vector according to claim 5 or 6, wherein the nucleotide sequence of the plant genetic transformation screening vector is represented by SEQ ID No. 3.

8. Use of the plant transgenic selection expression cassette of any one of claims 1 to 4 or the plant genetic transformation selection vector of any one of claims 5 to 7 for genetic transformation of plants, for preparing transgenic plants or for conferring high herbicide resistance to plants.

9. The application according to claim 8, wherein the application comprises: after the plant transgenic screening expression cassette or the plant genetic transformation screening vector is transferred into plant callus, a screening culture medium containing herbicide is adopted for resistance screening in a screening stage;

preferably, the screening culture medium contains 0.25-1 mu mol/L bispyribac-sodium, 200-1000 mu g/L imazapyr or 300-1000 mu g/L imazethapyr.

10. The application according to claim 8 or 9, characterized in that it comprises: in the differentiation stage after the plant transgenic screening expression cassette or the plant genetic transformation screening carrier is transferred into the plant callus, a differentiation culture medium containing herbicide is adopted for differentiation culture;

preferably, the differentiation medium contains 0.05-1 mu mol/L bispyribac-sodium, 25-1000 mu g/L imazapyr or 25-1000 mu g/L imazethapyr;

and/or the presence of a gas in the gas,

the application comprises the following steps: in the rooting stage after the plant transgenic screening expression box or the plant genetic transformation screening vector is transferred into the plant callus, a rooting culture medium containing herbicide is adopted for rooting culture;

preferably, the rooting medium contains 0.05-10 mu mol/L bispyribac-sodium, 25-1000 mu g/L imazapyr or 25-1000 mu g/L imazethapyr.

Technical Field

The invention relates to the technical field of agricultural biology, in particular to a plant transgenic screening expression cassette, a plant transgenic screening vector containing the expression cassette and application of the plant transgenic screening vector.

Background

With the rapid development of genetic engineering and molecular biology techniques, the application range of transgenic technology is more and more extensive. The transgenic technology has a plurality of advantages, such as widening available gene resources and creating new germplasm resources; can carry out directional and fixed-point variation and selection on plant physical traits; provides a new way for cultivating high-yield, high-quality and high-resistance fine varieties and the like. Transgenic crops are approved to be commercially planted in 1996, and 23 transgenic crops are totally approved for commercial production in 2017 worldwide, and the transgenic crops relate to more than 16 types of target traits including insect resistance, herbicide resistance, disease resistance, fertility change, quality improvement and the like. According to statistics, transgenic crops such as soybeans, corns, cottons, rapes and the like mainly resisting herbicides and insects are planted in 26 countries and regions in the world in 2017, and the area reaches 1.9 hundred million hectares. The large-area popularization of the transgenic crops makes great contribution to the global agricultural production.

At present, with the continuous popularization of transgenic products, people tend to be objective and positive in attitude of the transgenic products, but the controversial effects on the transgenic products are still serious, and the potential safety risk problems of the transgenic products are still generally concerned, so that the transgenic products need to be strictly controlled and supervised. One of the key issues for transgenic crops is the potential risk that a selectable marker may pose, and its resistance to target organisms, allergenicity of the expressed protein, and its effects on the nutrients, natural toxins and anti-nutrient content of the recipient crop itself are unknown after transfer into the plant. The most used at present are antibiotic and herbicide screening markers, such as hygromycin-HPT screening system, kanamycin-NPTII screening system, glufosinate-Bar screening system, etc. Most of these selection markers are foreign genes derived from bacteria, etc., which is also one of the causes of public concern. The risks and concerns posed by foreign genes can be eliminated if plants can be re-transformed with genes endogenous to the plant as selectable markers.

Acetolactate synthase (ALS; EC2.2.1.6) is a key enzyme catalyzing the initiation step of the synthesis of branched-chain amino acids (isoleucine, leucine and valine) in plants (Herrera-Estralla L, Block M D, M essens E, et al, nucleic genes as minor selectable markers in plant cells, the EM BO Journal,1983,2(6): 987. 995.), is a plant-specific enzyme which is absent in animals, and therefore can block the biosynthesis of branched-chain amino acids by inhibiting the ALS enzyme, thereby achieving the purpose of removing plant weeds, and is harmless to humans and animals. Currently, various herbicides such as sulfonylurea, imidazolinone, pyrimidinylsalicylic acid and sulfonamide herbicides, etc. have been developed and applied for the enzyme. Mutations at a certain site in the amino acid sequence of ALS may cause structural changes in the ALS protein, resulting in the failure of ALS inhibitors to bind ALS, rendering ALS less sensitive to herbicides and thus resistant to the relevant herbicides (Tan S, Evans RR, Dahm M L, et al. Imidazolinone-tolerant crops: history, currentstatus and future. Pest Management Science,2005,61 (3): 246-.

Disclosure of Invention

The invention aims to solve the problem of potential safety risk caused by using an exogenous screening marker in the current genetic transformation of a gene, and provides a vector for applying a plant endogenous gene as a screening marker to genetic transformation screening of a plant.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the invention firstly provides a plant transgenic screening expression cassette, which takes a plant endogenous gene as a screening marker, wherein the plant endogenous gene is ALS mutant gene.

Preferably, the ALS mutant gene is a rice ALS mutant gene.

More preferably, the nucleotide sequence of the rice ALS mutant gene (ALSm1) is shown as SEQ ID NO. 1.

The invention discovers that the ALS mutant gene with the sequence shown as SEQ ID NO.1 is more suitable to be used as a screening marker, and can more efficiently play the function of the screening marker in the process of plant gene transformation.

The plant transgene screening expression cassette further comprises a promoter and a terminator for initiating and terminating transcription of the ALS mutant gene.

The promoter may be a plant constitutive promoter or a plant tissue specific promoter.

Wherein, the plant constitutive promoter can be a rice ALS gene promoter, a Ubi promoter or an Actin promoter of rice or corn; the plant tissue specific promoter may be a Rubisco small subunit promoter or Cab promoter.

Preferably, the promoter is a rice ALS gene promoter.

The terminator may be a DNA sequence that can terminate gene transcription in plants, including the rice ALS gene terminator, Ubi terminator, and the like.

Preferably, the terminator is a rice Ubi terminator.

The invention discovers that the transcription of the ALS mutant gene of the rice can be well controlled by adopting the ALS gene promoter of the rice and the Ubi terminator to be matched and regulated, so that the high-efficiency, stable and proper expression of the ALS mutant gene can be well controlled, and the ALS gene mutant can better exert the function of a screening marker.

As a preferred scheme of the invention, the nucleotide sequence of the plant transgenic screening expression cassette is shown as SEQ ID No. 2.

The invention provides application of the ALS mutant gene as a screening marker for plant genetic transformation.

Based on the plant transgenic screening expression cassette, the invention further provides a plant genetic transformation screening vector, which contains the plant transgenic screening expression cassette.

Preferably, the plant genetic transformation screening vector is a plant binary expression vector, and can be used for genetic transformation by cloning other expression cassettes into the vector; the other expression cassettes refer to expression cassettes other than the plant transgene screening expression cassette, and include but are not limited to fluorescent protein expression cassettes, GUS reporter gene expression cassettes, insect-resistant expression cassettes, herbicide-resistant expression cassettes and the like.

More preferably, the plant genetic transformation screening vector of the present invention further comprises a series of multiple cloning sites such as AvrII, PmlI, SnaBI, AloI, HindIII, etc. to facilitate subsequent cloning of the gene of interest into it.

As a preferred scheme of the invention, the plant genetic transformation screening vector provided by the invention is pCALSm1, and the nucleotide sequence of the vector is shown as SEQ ID NO. 3.

The plant genetic transformation screening vector can be prepared by the following method:

(1) constructing a plant transgenic screening expression cassette: placing ALS mutant gene with a sequence shown as SEQ ID NO.1 into the gene under the drive of a promoter ALSpro of the gene to express, and terminating expression by a rice Ubi terminator at the downstream of the ALS mutant gene to obtain a plant transgenic screening expression cassette with a sequence shown as SEQ ID NO. 2;

(2) the plant transgenic screening expression cassette is connected with a plant binary expression vector pC0310 to obtain the plant genetic transformation screening vector.

The invention also provides application of the plant transgenic screening expression cassette or the plant genetic transformation screening vector in plant genetic transformation, preparation of transgenic plants or making plants have high herbicide resistance.

The herbicide of the invention includes but is not limited to imidazolinone, pyrimidine salicylic acid, sulfonylurea or sulfonamide herbicides. The application specifically comprises the following steps: after the plant transgenic screening expression cassette or the plant genetic transformation screening carrier is transferred into the plant callus, the callus is inoculated into a screening culture medium added with herbicide in the screening stage for resistance screening.

The herbicide may be a pyrimidinylsalicylate or imidazolinone herbicide, for example: bispyribac-sodium, imazapyr or imazethapyr.

Preferably, the screening culture medium contains 0.25-1 mu mol/L bispyribac-sodium, 200-1000 mu g/L imazapyr or 300-1000 mu g/L imazethapyr.

The screening culture medium preferably contains 0.3-0.4 mu mol/L bispyribac-sodium, and more preferably contains 0.4 mu mol/L bispyribac-sodium, so that good screening efficiency can be obtained; or preferably comprises 200-500 mu g/L of imazapyr, and more preferably comprises 250-300 mu g/L of imazapyr, so that good screening efficiency can be obtained; or preferably 300-500. mu.g/L imazethapyr, more preferably 400-500. mu.g/L imazethapyr, can obtain good screening efficiency.

Further, the application specifically comprises: and in the differentiation stage after the plant transgenic screening expression cassette or the plant genetic transformation screening vector is transferred into the plant callus, performing differentiation culture on the positive callus obtained from the screening culture medium by adopting a differentiation culture medium containing herbicide.

The herbicide may be a pyrimidinylsalicylate or imidazolinone herbicide, for example: bispyribac-sodium, imazapyr or imazethapyr.

Preferably, the differentiation medium contains 0.05-1 mu mol/L bispyribac-sodium, 25-1000 mu g/L imazapyr or 25-1000 mu g/L imazethapyr.

The differentiation medium preferably contains 0.05-0.25 mu mol/L bispyribac-sodium, and more preferably contains 0.1 mu mol/L bispyribac-sodium to obtain good differentiation efficiency; or preferably 30-100. mu.g/L of imazapyr, more preferably 50. mu.g/L of imazapyr, to achieve good differentiation efficiency; or preferably contains 30-100 mu g/L imazethapyr, more preferably contains 50 mu g/L imazethapyr, so that good differentiation efficiency can be obtained, and the non-transgenic callus can be effectively inhibited from differentiating into seedlings.

Further, the application specifically comprises: and in the rooting stage after the plant transgenic screening expression box or the plant genetic transformation screening vector is transferred into the plant callus, carrying out rooting culture on the positive seedling obtained by differentiation culture by adopting a rooting culture medium containing herbicide.

The herbicide may be a pyrimidinylsalicylate or imidazolinone herbicide, for example: bispyribac-sodium, imazapyr or imazethapyr.

Preferably, the rooting medium contains 0.05-10 mu mol/L bispyribac-sodium, 25-1000 mu g/L imazapyr or 25-1000 mu g/L imazethapyr.

The rooting culture medium preferably contains 0.05-5 mu mol/L bispyribac-sodium, and more preferably contains 0.1 mu mol/L bispyribac-sodium to obtain good rooting efficiency; or preferably 50-100 mug/L of imazapyr, more preferably 50 mug/L of imazapyr, to obtain good rooting efficiency; or preferably contains 50-100 mu g/L imazethapyr, more preferably contains 50 mu g/L imazethapyr, so that good rooting efficiency can be obtained, and simultaneously, the rooting of the non-transgenic differentiated seedling can be effectively inhibited.

Plants of the invention include, but are not limited to, rice, maize, wheat, soybean, sorghum, peanut, sesame, cotton, linseed, grain, oat, rapeseed, barley, rye, millet, tobacco, highland barley, arabidopsis, and the like. Preferably, the plant is rice, maize, wheat, soybean, sorghum, peanut or millet.

Taking rice as an example, other plant transgenic methods can refer to rice:

1) induction: after rice seeds are shelled and disinfected, mature embryos are inoculated in an induction culture medium to induce embryonic callus, and dark culture is carried out for 30-50 days at the temperature of 27 ℃;

2) infection: separating the callus obtained in the step 1) from endosperm and buds, inoculating the callus into a suspension of agrobacterium carrying the plant genetic transformation screening vector (suspending the agrobacterium carrying the plant genetic transformation screening vector in a suspension culture medium) for infection, standing for 30 minutes at room temperature, and then airing for later use;

3) co-culturing: transferring the dried callus into a co-culture medium, and performing dark culture at 22 ℃ for 3 days until thalli appear on the surface of the callus;

4) screening: cleaning the co-cultured callus, inoculating to a screening culture medium containing bispyribac-sodium, imazapyr or imazethapyr, performing dark culture at 27 deg.C for 30-50 days, and screening for resistance;

5) differentiation: inoculating the obtained resistant callus onto a differentiation culture medium added with bispyribac-sodium, imazapyr or imazethapyr, and performing illumination culture at 27 ℃ for 25-40 days until seedlings are differentiated;

6) rooting: inoculating the seedling to a rooting culture medium added with bispyribac-sodium, imazapyr or imazethapyr for rooting, performing illumination culture at 30 ℃ for 10-20 days, performing PCR detection, and selecting the plant which is detected to be positive for planting;

the specific formula of the culture medium is as follows:

the induction medium is N6D medium, which is a medium taking N6 medium as a basic medium, and contains 3mg/L of 2, 4-dichlorophenoxyacetic acid (2,4-D), 0.3-0.6g/L of hydrolyzed Casein (CH), 0.3-0.5g/L of proline (Pro), 30g/L of sucrose, 3g/L of plant gel (Phytagel) and 5.9 of pH value;

the suspension culture medium is an N6-AA culture medium, which takes an N6 culture medium AS a basic culture medium, and contains 2, 4-dichlorophenoxyacetic acid (2,4-D) with the concentration of 2mg/L, hydrolyzed Casein (CH) with the concentration of 0.3-0.6g/L, proline (Pro) with the concentration of 0.3-0.5g/L, sucrose with the concentration of 20g/L, glucose with the concentration of 10g/L, Acetosyringone (AS) with the concentration of 100-;

the co-culture medium is N6-AS culture medium, which takes N6 culture medium AS basic culture medium, and contains 2, 4-dichlorophenoxyacetic acid (2,4-D) with the concentration of 2mg/L, hydrolyzed Casein (CH) with the concentration of 0.3-0.6g/L, proline (Pro) with the concentration of 0.3-0.5g/L, sucrose with the concentration of 20g/L, glucose with the concentration of 10g/L, Acetosyringone (AS) with the concentration of 100-;

the screening culture medium is N6S culture medium, which takes N6 culture medium as basic culture medium, and contains 2, 4-dichlorophenoxyacetic acid (2,4-D) with the concentration of 2-3mg/L, hydrolyzed Casein (CH) with the concentration of 0.6-1g/L, proline (Pro) with the concentration of 0.5-1.5g/L, sucrose with the concentration of 30g/L, plant gel (Phytagel) with the concentration of 3g/L, and cephalosporin (Cn) with the concentration of 500 mg/L. The concentration of the screening agent bispyribac-sodium in the screening culture medium is 0.25-1 mu mol/L, or the concentration of the imazapyr is 200-;

the differentiation culture medium is MSRe culture medium which takes MS culture medium as basic culture medium, and contains 1-2mg/L of Kinetin (KT), 0.5-2mg/L of alpha-naphthylacetic acid (NAA), 20-40g/L of sorbitol, 30g/L of sucrose and 3g/L of plant gel (Phytagel). A culture medium with the concentration of the screening agent bispyribac-sodium of 0.05-1 mu mol/L, or the concentration of imazapyr of 25-1000 mu g/L, or the concentration of imazethapyr of 25-1000 mu g/L and the pH value of 5.9 in a differentiation culture medium;

the rooting medium is 1/2MSR medium, which is 1/2MS medium as basic medium, and contains 20g/L sucrose, 0.5-1mg/L paclobutrazol and 3g/L plant gel (Phytagel). A culture medium with the concentration of the screening agent bispyribac-sodium of 0.05-10 mu mol/L, the concentration of imazapyr of 25-1000 mu g/L or the concentration of imazethapyr of 25-1000 mu g/L and the pH value of 5.8 in the rooting culture medium;

the beneficial effects of the invention at least comprise: the invention provides a plant transgenic screening expression box using a rice ALS mutant gene as a screening marker, a plant genetic transformation screening vector and a corresponding gene transformation screening method. The plant genetic transformation screening vector can be used as a transgenic screening vector to be added with other functional elements for plant gene transformation. The screening marker of the invention is a plant endogenous gene, and exogenous screening marker genes such as a bacterial source and the like are not introduced in the transgenic process, so that the method not only enriches the screening method of the plant transgene, but also can effectively reduce the potential safety risk of the transgenic plant caused by the exogenous gene and the public worry about the safety of the transgenic plant, is beneficial to the commercial application of the transgenic plant, and has good market value and social benefit.

Drawings

FIG. 1 is an electrophoretogram of pCALSm1 vector digested with NcoI and PstI in example 1 of the present invention; wherein, M is Marker, CK is pCALSm1 recombinant plasmid which is not cut by enzyme, 1-5 is pCALSm1 recombinant plasmid which is cut by enzyme, and a fragment with the size of about 2.2kb can be cut out.

FIG. 2 shows the result of PCR detection electrophoresis of Agrobacterium transformed in example 2 of the present invention; wherein, M is Marker, 1 is pCALSm1 recombinant plasmid positive control, and 2-6 are pCALSm1 recombinant plasmid agrobacterium monoclonal bacteria liquid samples.

FIG. 3 is a map of the pC0310 vector in example 1 of the present invention.

FIG. 4 is a map of the pCALSm1 vector in example 1 of the present invention.

FIG. 5 is a comparison chart of critical concentration tests of bispyribac-sodium in seedling stage of common rice in example 3 of the present invention; wherein, the left side of the seedling pot is 9311, the right side is ZH11, wherein 0 represents that bispyribac-sodium is not added, 10 x represents 300mg/L, and 20 x is 600 mg/L.

FIG. 6 is a test chart of critical concentration of imazapyr in the seedling stage of ordinary rice in example 3 of the present invention; wherein 0.2X represents 19.6mg/L, and 0.5X, 1X, 2X and 5X are corresponding multiples respectively; the seedlings of the seedling pot are ZH 11.

FIG. 7 shows critical concentration test of imazethapyr in seedling stage of ordinary rice in example 3 of the present invention; wherein 5X represents 375mg/L, 20X, 50X, 100X, 300X are the corresponding multiples respectively; the seedlings of the seedling pot are ZH 11.

FIG. 8 shows the results of critical concentration test of rice calli against bispyribac-sodium in example 4 of the present invention; wherein, N6 is the result of the callus screening for 40 days on the culture without adding screening pressure, and N6+ is the screening pressure of adding 0.25, 0.5, 1 and 2 mu mol/L bispyribac-sodium respectively.

FIG. 9 shows the results of callus screening with bispyribac-sodium screening medium in example 8 of the present invention for 40 days.

FIG. 10 shows the results of differentiation of callus 30d by adding bispyribac-sodium, imazapyr or imazethapyr to the differentiation medium in examples 14, 20 and 28 of the present invention; wherein, 1 and 2 are differentiation culture with bispyribac-sodium (0.1 mu mol/L), 3 and 4 are differentiation culture with imazapyr (50 mu g/L), and 5 and 6 are differentiation culture with imazethapyr (50 mu g/L); wherein 1, 3 and 5 are non-transgenic calli and 2,4 and 6 are transgenic positive calli.

FIG. 11 shows the results of rooting 15 days in rooting medium of examples 37, 47 and 55 according to the present invention with bispyribac-sodium, imazapyr or imazethapyr; wherein, 1 and 2 are rooting culture with bispyribac-sodium (0.1 mu mol/L), 3 and 4 are rooting culture with imazapyr (50 mu g/L), and 5 and 6 are rooting culture with imazethapyr (50 mu g/L); wherein, 1, 3 and 5 are used for rooting of non-transgenic differentiated seedlings, and 2,4 and 6 are used for rooting of transgenic positive differentiated seedlings.

FIG. 12 is a PCR detection electrophoretogram of transgenic sample plants in example 63 of the present invention; wherein M is Marker, 1 is H₂O and 2 are genome DNA of a ZH11 non-transgenic plant, 3 is a plasmid positive control, and 5-24 are genome DNA of a transgenic plant obtained by screening.

FIG. 13 shows the results of the herbicide resistance test of T0 transgenic line in example 64 of the present invention; wherein, a-d: after the bispyribac-sodium screened plant line is transplanted, 60mg/m of bispyribac-sodium is sprayed²The results of bispyribac-sodium are shown in the specification, a-d are respectively before spraying, 7 days, 14 days and 21 days after spraying, arrows in the figure show that wild type control ZH11 is completely dead after spraying for 14 days, e and f are respectively 0.5 × (49mg/L) imazapyr after a imazapyr screening line is transplanted, e is before spraying, f is after spraying for 21 days, g-h is after spraying for 21 days, 5 × (375mg/L) imazethapyr is sprayed after an imazethapyr screening line, g is before spraying, h is after spraying for 21 days, wherein ZH11 is wild type control, 9311 and MH63 are common cultivation rice control, and 47-1, 48-2, 61-1 and 63-1 are transgenic lines.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.