AU2021101533A4 - APPLICATION OF OsbZIP62-VP64 FUSION EXPRESSION IN IMPROVING AGRONOMIC TRAITS OF RICE - Google Patents

Abstract

The present disclosure discloses application of OsbZP62-VP64 fusion expression in improving agronomic traits of rice. The present disclosure conducts fusion recombination (OsbZIP62-VP64 and OsbZIP62V) of a transcription activation domain variant VP64 derived from a herpes virus and having an enhanced rice gene transcription activity with an OsbZP62 gene in rice. Over-expression of the OsbZIP62V gene can improve agronomic traits of rice such as plant height, panicle length, the number of grains per panicle, and the number of primary/secondary rachis branches. The rice gene OsbZIP62V of the present disclosure has obvious positive effects on agronomic traits of rice, can be used for molecular breeding of rice, and plays an important role in enriching germplasm resources of rice, creating new germplasm, and even shortening a breeding process. 1/3 VP64 pBCV pUbi FIG. 1 300 p 200 100 0 0 0 FIG. 2

Description

1/3

VP64

pBCV pUbi

FIG. 1

300

p 200

100

0

0 0 FIG. 2

APPLICATION OF OsbZIP62-VP64 FUSION EXPRESSION IN IMPROVING

AGRONOMIC TRAITS OF RICE TECHNICAL FIELD

The present disclosure relates to a gene related to important agronomic traits of rice,

particularly to application of OsbZIP62-VP64 fusion expression in improving

agronomic traits of rice, and belongs to the field of genetic engineering.

BACKGROUND

Rice is an important food crop and provides food security for more than 50% of the

world's population. In recent years, with more human activities, climatic disasters

occurred more frequently. The frequent climatic disasters severely affect

physiological metabolism of rice, thus affect physical and chemical properties of rice,

such as plant height, panicle length and yield, and severely restrict a long-term goal

for breeding crops with high and stable yield. Therefore, it is of great importance to

have an in-depth study of physiological and molecular mechanisms of rice plant

height and other agronomic traits for cultivating new rice varieties with high and

stable yield and ensuring production safety of food.

Since entering the 21st century, genomics, proteomics and other disciplines and a

gene editing technology develop rapidly. In order to deal with aging of farmers,

reduction of arable land, deterioration of the environment and increased demand for

food quality, a concept of design breeding is proposed. In recent years, with

breakthroughs of breeding theories and technologies, a breeding technology, namely

molecular design breeding has become a reality. The development of the breeding

technology is the core content of green super rice. Cloning and identification of

important agronomic traits is a prerequisite and basis for molecular design breeding.

Only when a molecular mechanism of formation of crop traits is analyzed at a

genetic level, accurate design and implementation are conducted during a breeding

process. Excellent genes are combined and a chain burden between the excellent

genes and unfavorable genes is broken, thereby achieving targeted design breeding

and greatly improving breeding efficiency. The improvement of plant type in

agronomic traits is always one of core indicators of rice varieties with high and stable

yield. Plant type of rice directly affects the number of effective panicles and grains

per panicle, and is a core element that determines yield. Therefore, a molecular

design breeding concept is used to breed rice varieties with high and stable yield and

a core lies in digging out key genes that affect plant type and performing detailed

functional analysis. It has been reported that some genes are involved in regulation

of plant type of rice, such as GRAS family protein single tiller genes MOCI and

MOC3 playing an important role in regulating plant type of rice and a transcription

factor IPAI gene containing an SBP-box domain. However, plant type is mostly a

result of a common action of multiple genes and multiple mechanisms and has fewer

major genes. Therefore, how to improve an effect of a single gene is the key to

breeding. The selection of different types of promoters is one of commonly used

methods to enhance an effect of gene expression. At present, many promoters

suitable for plants have been isolated from animals/plants, viruses and

microorganisms. According to action modes and functions, promoters can be divided

into three types: constitutive promoters, inducible promoters and tissue-specific

promoters. However, expression effects of many genes are still unsatisfactory, so

that their related mechanisms need to be studied in depth.

SUMMARY

The present disclosure is intended to provide application of OsbZP62-VP64 fusion

expression in improving agronomic traits of rice. The present disclosure finds an

OsbZIP62V gene related to agronomic traits of rice and a protein encoded by the

OsbZIP62V gene. Over-expression of the OsbZIP62V gene can improve agronomic

traits of plant height, panicle length, the number of grains per panicle, and the

number of primary/secondary rachis branches of a transgenic plant.

The present disclosure fuses a complete DNA fragment VP64 with a complete DNA

fragment OsbZIP62 isolated and cloned from rice to form an OsbZ/P62V fusion

protein fused with a transcription activation domain.

The present disclosure isolates and uses a DNA fragment containing an OsbZP62V

gene and the gene can affect important agronomic traits such as plant height and

panicle length of rice.

The objectives of the present disclosure are achieved by the following technical

solutions.

The present disclosure discloses application of an OsbZIP62V gene or a protein

encoded by the OsbZP62V gene in improving important agronomic traits of rice.

As an implementation of the present disclosure, the important agronomic traits of rice

may include plant height, panicle length, the number of grains per panicle, and the

number of primary/secondary rachis branches of rice.

As an implementation of the present disclosure, the application may include

constructing an OsbZIP62V over-expression vector and introducing the vector into a

plant cell to obtain a transgenic plant.

As an implementation of the present disclosure, the OsbZIP62 gene in rice is

recombinantly cloned into an over-expression vector containing a transcription

activation domain variant VP64 to obtain the OsbZP62V over-expression vector.

As an implementation of the present disclosure, the transcription activation domain

variant VP64 and OsbZIP62 gene in rice are subjected to fusion recombination, and

an amplified product fragment is cloned into an over-expression vector to obtain the

OsbZIP62V over-expression vector.

As an implementation of the present disclosure, the transcription activation domain

variant VP64 is derived from a herpes virus.

As an implementation of the present disclosure, a Ti plasmid or a plant virus vector is

used for constructing the OsbZP62V over-expression vector.

As an implementation of the present disclosure, an sequence of the OsbZP62V

gene is a DNA sequence shown in SEQ ID NO: 1, or a DNA sequence that is at least

% homologous to SEQ ID NO: 1, or a sub-fragment with a function equivalent to

the sequence shown in SEQ ID NO: 1.

As an implementation of the present disclosure, an amino acid sequence of the

protein encoded by the OsbZIP62V gene is shown in SEQ ID NO: 2 in the sequence

listing, or a homologous sequence, a conservative variant, an allelic variant, a

natural mutant or an induced mutant of a sequence shown in SEQ ID NO: 2.

The present disclosure also provides a recombinant vector and a plant transformant

encoded by an OsbZIP62V gene, a Ti plasmid or a plant virus vector is used for

constructing the recombinant vector, and a host of the plant transformant is rice.

A polymerase chain reaction (PCR) technology can be used to amplify the

OsbZIP62V gene and any section of DNA of interest or a section of DNA

homologous thereto from genome, mRNA or cDNA. By using the PCR technology, the OsbZIP62V gene can be isolated. The sequence is ligated with any vector that can guide expression of a foreign gene in a plant to transform a plant to obtain a transgenic plant with affected agronomic traits.

An expression vector carrying an OsbZIP62V gene is provided by the present

disclosure and can be introduced into a plant cell by using a Ti plasmid, a plant virus

vector, microinjection and electroporation and other conventional biotechnological

methods.

A host transformed by the OsbZIP62V gene expression vector of the present

disclosure is a variety of plants including rice.

Through cloning, separation and fusion of genes in rice and an evaluation of

influence on agronomic traits of rice, the present disclosure provides a new

recombined rice DNA fragment, which contains a 975 bp encoding gene OsbZP62V.

The gene significantly improves agronomic traits of rice such as plant height, panicle

length, the number of grains per panicle, and the number of primary/secondary

rachis branches.

The present disclosure has the following beneficial effects compared with the prior

art.

1) The present disclosure conducts recombination (OsbZP62-VP64 and

OsbZIP62V) of a transcription activation VP64 and an OsbZP62 transcription factor.

Over-expression of the OsbZIP62V gene can improve agronomic traits of rice such

as plant height, panicle length, the number of grains per panicle, and the number of

primary/secondary rachis branches;

2) The rice gene of the present disclosure has a significant influence on agronomic

traits and can be applied to plant molecular breeding; and

3) The present disclosure can be used to study a molecular method for obtaining a

transgenic plant by using the gene for genetic transformation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a structural diagram of OsbZP62 (OsbZP62V) containing a VP64

activation domain;

FIG. 2 shows detection of an expression level of an OsbZP62V gene over

expression transgenic rice plant;

FIG. 3 shows a comparison analysis of plant height between OsbZP62V gene over

expression transgenic rice and wild-type rice;

FIG. 4 shows a comparison analysis of the number of rachis branches between

OsbZIP62V gene over-expression transgenic rice and wild-type rice;

FIG. 5 is shows a comparison analysis of panicle length between OsbZP62V gene

over-expression transgenic rice and wild-type rice;.

DETAILED DESCRIPTION

The following examples further describe the disclosure, but the examples are only intended to illustrate the disclosure, rather than limiting the disclosure. Modifications or substitutions made to methods, steps or conditions of the present disclosure are all belong to the scope of the present disclosure, without departing from the spirit and essence of the present disclosure.

The experimental methods in the following examples which are not specified with specific conditions are generally carried out under conventional conditions for example, conditions disclosed in Molecular Cloning: Experiment Guide (Sambrook et al., New York: Cold Spring Harbor Laboratory Press, 1989) or conditions recommended by manufacturers.

Example 1 Cloning of OsbZIP62 gene of rice

A plasmid OsDTHI constructed and stored in the laboratory was used as a template, an upstream primer OsbZIP62F (5'-ACAGCCCTGGAACATTGGCC-3' SEQ ID NO.3) and a downstream primer OsbZIP62R (5' TAGTTAAGAAAAGAGTTCGTCG-3' SEQ ID NO.4) were used to amplify an OsbZIP62 target fragment, gel recovery was conducted, the OsbZIP62 target fragment was connected to a pEASY-Blunt vector, sequencing was conducted after identification and sequencing results were confirmed by BLAST comparison. The results showed that a CDS sequence of the OsbZIP62 gene of rice was 825 bp.

Example 2 Transformation of rice by over-expression vector containing fusion gene OsbZIP62V of rice

1. A GATEWAY recombinant cloning technology was used to construct an over expression vector containing an OsbZP62 gene and a structure of the vector was shown in FIG. 1:

The pEasy-Blunt vector containing the OsbZIP62 gene obtained in Example 1 was used as a template, and a forward primer F: 5' AAAAAGCAGGCTATGGGAGTCCACGCA-3'SEQ ID NO. 5 and a reverse primer R: '-AGAAAGCTGGGTTTAGAAAGAGGC-3'SEQ ID NO. 6 were used to perform the first round of PCR amplification. Then universal primers attB1 adapter: 5' GGGGACAAGTTTGTACAAAAAAGCAGGCT-3' SEQ ID NO. 7 and attB2 adapter: '-GGGGACCACTTTGTACAAGAAAGCTGGGT-3' SEQ ID NO. 8 were used to perform the second round of PCR amplification, after an amplified product was recovered and purified, the amplified product fragment was cloned into an entry vector pDONR by a BP reaction, positive clones were screened, the target gene was cloned into an over-expression vector pBCV by a LR reaction. Specific processes were as follows:

(1) The first round of PCR amplification

A 20-pL reaction system was as shown in Table 1:

Table 1

Reaction Component Volume (pL)

ddH20 13.8 10xbuffer 2 dNTPs 1

Primer F 1

Primer R 1

Plasmid 1

Taq 0.2

An amplification procedure: pre-denaturation at 98C for 2 min, and a total of 10 cycles of 98C for 15 s, 60°C for 30 s and 72C for 2 min.

(2) The second round of PCR amplification

10 pL of the above PCR product was used as a template and added to a 40-pL reaction system prepared below for PCR. Table 2

Reaction Component Volume (pL)

ddH20 19.5

1Oxbuffer 4

dNTPs 2

Primer F 2

Primer R 2

PCR product 10

Taq 0.5

An amplification procedure: pre-denaturation at 98C for 1 min, and a total of 10 cycles of 98C for 15 s, 45C for 30 s and 720C for 2 min and a total of 25 cycles of 980C for 15 s, 550C for 30 s and 720C for 2 min.

After the reaction was ended, 5 pL of the PCR product was subjected to an electrophoresis detection.

(3) Recovery of PCR product

An ordinary agarose gel DNA recovery kit was used for purification and recovery.

(4) BP recombination reaction

A recombination reaction system can be prepared and mixed at room temperature and a recombination reaction was carried out in a0.5-mL centrifuge tube. The reaction system was as shown in Table 3: Table 3

Reagent Volume

attB-PCR product(>1Ong) 3pL

pDONR vector (150 ng/pL) 1pL

5xBP Clonase Clonase enzyme mix 1pL

A warm bath was conducted at 250C for about 16 hours and a BP reaction solution transformed competent escherichia coli cells. Recombinant escherichia coli must be grown on plates containing gentamicin. Single colonies were picked for a PCR verification and plasmid were extracted.

(5) LR recombination reaction

A recombination reaction system can be prepared at room temperature and a recombination reaction was carried out in a 0.5-mL centrifuge tube. The reaction system was as shown in Table 4:

Table 4

Reagent Volume

Entry clone (50-150 ng) 3 pL

Destination vector (150 ng/pL) 1 pL

5xLR Clonase enzyme mix 1 pL

A warm bath was conducted at 250C for about 16 hours and a reaction solution transformed competent escherichia coli cells. The transformed escherichia coli solution was spread on plates containing kanamycin for growing. Single colonies were picked, A PCR verification was conducted, sequencing was conducted, sequencing result was compared with a cDNA sequence of the gene to confirm whether the sequence was correct or not, and plasmids were extracted to transform agrobacterium tumefaciens EHA105.

2. Transformation of agrobacterium tumefaciens

(1) Preparation of competent agrobacterium tumefaciens (EHA105) cells:

When an agrobacterium tumefaciens solution was cultured at 280C to OD600=0.5, the bacteria were collected by centrifugation at 4C and resuspended in 500 pL of 0.1 mol/L CaCl2 in an ice bath, centrifugation was conducted after the ice bath for 30 min, a supernatant was removed and the bacteria were resuspended in 100 pL of 0.1 mol/L CaCl2 in an ice bath and stored at 4C.

(2) Transformation of agrobacterium tumefaciens (freeze-thaw method):

5 pL of plant expression vector plasmid DNA was added to the competent agrobacterium tumefaciens cells (100 pL) and mixed gently, after 30 min of an ice water bath, and a quick-freezing cold shock was conducted in liquid nitrogen for 2 min; 400-800 pL of a YEP medium (containing kanamycin (Kan), 50 mg/L) was added; shaking culture at 28C and 200 r/min was conducted for 3-5 h; a room temperature centrifugation was conducted (5,000 r/min, 5min), 100 pL of a supernatant of resuspended bacteria was reserved and spread on a LB solid medium (containing Kan, 50 mg/L), inverted culture was conducted at 28C for 2 days until colonies of a suitable size grew, and single clones were picked for a PCR detection to obtain a positive strain.

3. Callus induction: seeds were rinsed with sterile water for 15-20 min and disinfected with 75% ethanol for 1 min, and disinfected with a sodium hypochlorite solution (at 1.5% effective concentration) in a shaking manner for 20 min. The seeds were rinsed with sterile water for 5 times. The washed seeds were blotted dry with absorbent paper and inoculated in a callus induction medium, and cultured in the dark at 250C for 2 weeks.

The callus induction medium was prepared by the following steps: an induction medium in Table 5 was used, 0.3 g of proline, 0.6 g of hydrolyzed casease, 30 g of sucrose and 2.5 mL of 2,4-D (at a concentration of 1 mg/mL) were added, the components were prepared into 1 L of a solution, pH was adjusted to 5.9, 7 g of agar powder was added, and sterilization at a high temperature and pressure was conducted.

4. Subculture: an embryogenic callus was cut off, inoculated into a subculture medium and cultured in the dark at 25 0 C for 2 weeks.

The subculture medium was prepared by the following steps: a subculture medium in Table 5 was used, 0.5 g of proline, 0.6 g of hydrolyzed casease, 30 g of sucrose and 2 mL of 2,4-D (at a concentration of 1 mg/mL) were added, the components were prepared into 1 L of a solution, pH was adjusted to 5.9, 7 g of agar powder was added, and sterilization at a high temperature and pressure was conducted.

5. Agrobacterium tumefaciens infection and callus co-cultivation: agrobacterium tumefaciens was cultured and positive single colonies were picked and cultured at 28°C overnight in 1 mL of 50 mg/L YEP agrobacterium tumefaciens culture solution (containing antibiotics); and the above cultured material was added into 50 mL of an agrobacterium tumefaciens culture solution (containing antibiotics) to be cultured at 280C to OD600=0.6-1.0. The obtained agrobacterium tumefaciens solution was centrifuged, the collected bacteria were added into a suspension medium, and shake culture was conducted for 30 min to OD600=0.6-1.0. The callus was put into the suspension medium containing the agrobacterium tumefaciens solution and shake culture was conducted for about 20 min. The callus was air-dried on sterile filter paper, transferred to a co-cultivation medium, and cultured in the dark at 250C for 5 d.

The suspension medium was prepared by the following steps: a suspension medium in Table 5 was used, 0.08 g of hydrolyzed casease, 2 g of sucrose and 0.2 mL of 2,4-D (at a concentration of 1 mg/mL) were added, the components were prepared into 100 mL of a solution, pH was adjusted to 5.4, the solution was divided into two bottles (with 50 ml of each bottle) and sterilization at a high temperature and pressure was conducted. 1 mL of 50% glucose and 100 pL of AS (100 mM) were added before use.

The co-cultivation medium was prepared by the following steps: a co-cultivation medium in Table 5 was used, 0.8 g of hydrolyzed casease, 20 g of sucrose and 3.0 mL of 2,4-D (at a concentration of 1 mg/mL) were added, the components were prepared into 1 L of a solution, pH was adjusted to 5.6, 7 g of agar powder was added, and sterilization at a high temperature and pressure was conducted. 20 mL of % glucose and 1 mL of AS (100 mM) were added before use.

6. Screening culture: After the co-cultivation for 3 d, a good callus was selected, transferred to a screening medium, cultured in the dark at 250C for 2 weeks, and screened twice.

The screening medium was prepared by the following steps: a screening medium in Table 6 was used, 0.6 g of hydrolyzed casease, 30 g of sucrose and 2.5 mL of 2,4-D (at a concentration of 1 mg/mL) were added, the components were prepared into 1 L of a solution, pH was adjusted to 6.0, 7 g of agar powder was added, and sterilization at a high temperature and pressure was conducted. 1 mL of Hn and 1 mL of Cn (100 ppm) were added before use.

7. Differentiation culture: an embryogenic callus was picked, inoculated into a differentiation medium, and subjected to 16h/8h light and dark culture at 240C to induce differentiation buds (4-6 weeks).

The differentiation medium was prepared by the following steps: a differentiation medium in Table 6 was used, 2.0 mg/L of 6-BA, 2.0 mg/L of KT, 0.2 mg/L of NAA, 0.2 mg/L of IAA, 1.0 g of hydrolyzed casease and 30 g of sucrose were added, the components were prepared into 1 L of a solution, pH was adjusted to 6.0, 7 g of agar powder was added, and sterilization at a high temperature and pressure was conducted.

8. Rooting culture: when the buds grew to about 2 cm, and the young buds were cut off, inserted into a rooting medium, and subjected to 16h/8h light and dark culture at 250C to induce rooting.

The rooting medium was prepared by the following steps: a rooting medium in Table 6 was used, 30 g of sucrose was added, the components were prepared into 1 L of a solution, pH was adjusted to 5.8, 7 g of agar powder was added, and sterilization at a high temperature and pressure was conducted.

9. Cultivation of transformed plants: after a root system was developed, a mouth of a test tube was opened, sterile water was added for exercising seedlings for 2-3 d, then the plants were taken out, attached solid medium was washed away with sterile water, the plants were moved to the soil, shading and sheltering from wind were conducted at a beginning, and management cultivation in a conventional field or a greenho application was conducted after the plants were strong. Table 5 Components 1 of Basic Medium

Component N6 (mg/L) Induction and Suspension and Subculture Medium Co-cultivation (mg/L) Medium (mg/L)

Major Elements

KNO3 2830 2830 1415

KH2PO4 400 200 200

(NH4)2SO4 463 463 231.5

MgSO4-7H20 185 185 62.5

CaC12-2H20 166 166 83

Trace Elements

ZnSO4-7H20 1.5 1.5 0.75

MnSO4-4H20 4.4 4.4 2.2

H3B03 1.6 1.6 0.8

KI 0.8 0.8 0.4

Fe 2+-EDTA (ferric salt)

FeSO4-7H20 27.8 27.8 27.8

Na2EDTA-2H20 37.3 37.3 37.3

Vitamin (organic ingredients)

Glycine 2 2 2

Thiamine 1 1 1 HCL(VB1)

Pyridoxine 1 1 1 HCL(VB6)

Nicotinic acid 1 1 1

Inositol 100 100 100

Table 6 Components 2 of Basic Medium

Component MS (mg/L) Screening and Rooting Medium Differentiation (mg/L) Medium (mg/L)

Major Elements

KNO3 1900 1900 950 NH4NO3 1650 1650 825

KH2PO4 170 170 85

MgSO4-7H20 370 370 185

CaCl2-2H20 440 440 220

Trace Elements

ZnSO4-7H20 1.5 1.5 0.75

MnSO 4 -4H20 22.3 22.3 11.15

H3BO3 6.2 6.2 3.1

KI 0.83 0.83 0.415 CuS04-5H20 0.025 0.025 0.0125

Na2M04-2H20 0.25 0.25 0.125

CoC12-6H20 0.025 0.025 0.0125

Fe 2+-EDTA (ferric salt)

FeSO4-7H20 27.8 27.8 27.8

Na2EDTA-2H20 37.3 37.3 37.3

Vitamin (organic ingredients)

Glycine 2 2 2

Thiamine 1 1 1 HCL(VB1)

Pyridoxine 1 1 1 HCL(VB6)

Nicotinic acid 1 1 1

Inositol 100 100 100

10. Detection of expression level of target gene in over-expression positive plants

A RNA extraction and quantitative PCR method was shown in Example 1.

RNA of leaves of transgenic T3 generation plants was extracted. An expression level of the target gene in OsbZP62V over-expression plants was detected by quantitative PCR (FIG. 2). In the detected two transgenic plants, the expression level increased to varying degrees.

Example 3 Identification of agronomic traits of OsbZP62V over-expression transgenic rice

Seeds of OsbZP62V over-expression transgenic families were shelled and disinfected (treatment with 75% alcohol for 1 min, treatment with 1.5% NaCIO for 20 min and washing with sterile water for 4-5 times) and germinate on a 1/2MS medium containing 50 mg/L of hygromycin. A wild-type control was sown one day later on a 1/2MS medium without hygromycin. After 2-3 days of germination, seedlings with good germination and consistent growth were transplanted into the field and phenotypes were observed until the seedlings grew to a booting stage. Experiment results showed that the over-expression OsbZIP62V transgenic plants (OE11 and OE12) had higher plant height, more number of rachis branches, and longer panicle length than those of the wild-type control (WT) (FIG. 3-FIG. 5). The results showed that agronomic traits of rice were improved by an OsbZP62V over-expression gene. In conclusion, although the present disclosure has been described in detail by some cited specific examples, it is apparent to those skilled in the art that various changes or modifications may be made without departing from the spirit and scope of the present disclosure.

Claims (5)

1. An over-expression vector for improving agronomic traits of rice, comprising a OsbZIP62V gene.

2. The over-expression vector of claim 1, wherein the agronomic traits of rice comprise plant height, panicle length, the number of grains per panicle, and the number of primary/secondary rachis branches of rice.

3. A over-expression vectorfor constructing an OsbZP62V over-expression vector, wherein, the OsbZIP62 gene in rice is recombinantly cloned into an over-expression vector containing a transcription activation domain variant VP64 to obtain the OsbZIP62V over-expression vector; wherein, the transcription activation domain variant VP64 is derived from a herpes virus; wherein, a Ti plasmid or a plant virus vector is used for constructing the OsbZP62V over-expression vector.

4. The over-expression vector of claim 1 or 2, wherein, a sequence of the OsbZIP62V gene is a DNA sequence shown in SEQ ID NO: 1, or a DNA sequence that is at least 90% homologous to SEQ ID NO: 1, or a sub-fragment with a function equivalent to the sequence shown in SEQ ID NO: 1.

5. The over-expression vector of claim 1 or 2, wherein, an amino acid sequence of a protein encoded by the OsbZIP62V gene is shown in SEQ ID NO: 2 in the sequence listing, or a homologous sequence, a conservative variant, an allelic variant, a natural mutant or an induced mutant of a sequence shown in SEQ ID NO: 2.