A strong promoter cp09 specifically expressed in plant pollen and its application

Abstract

The invention discloses a strong promoter CP09 specifically expressed in plant pollen, a gene expression cassette containing CP09, a recombinant expression vector, a recombinant bacterium and a transgenic plant cell line, the strong promoter CP09 specifically expressed in plant pollen , gene expression cassettes, recombinant expression vectors, recombinant bacteria and transgenic plant cell lines can efficiently drive the specific expression of exogenous genes in plant pollen, and the expression level is precise, which can avoid the adverse effects caused by the continuous expression of target genes in other plant tissues. It can be used to create male sterile lines of plants, and has good application prospects in plant genetic engineering and utilization of heterosis. Moreover, the nucleotide sequence of the strong promoter CP09 specifically expressed in plant pollen disclosed by the present invention is relatively short, and its full length is only 409bp, which is easy to clone, can effectively reduce the difficulty and cost of cloning the promoter, and helps to improve the efficiency of recombination. The construction and transformation efficiency of the expression vector help to obtain its transgenic plants quickly.

Description

technical field

[0001] The invention relates to the field of plant genetic engineering and molecular biology, in particular to a strong promoter CP09 specifically expressed in plant pollen and its application. Background technique

[0002] The regulation of plant gene expression is mainly at the transcriptional level, which is coordinated by a variety of cis-acting elements and trans-acting factors. Promoters are important cis-acting elements that play a key role in transcriptional regulation. Promoters can be divided into three categories according to different expression modes, namely constitutive promoters, inducible promoters and tissue-specific promoters. , tubers, vascular bundles, flowers, pollen and seeds, etc.) to drive gene expression, to achieve timed and quantitative precise regulation of exogenous gene expression in plants, which can effectively reduce the energy loss or energy loss caused by the continuous expression of constitutive promoters in different part...

Examples

Embodiment 1

[0031] Example 1: Obtainment of a strong promoter CP09 specifically expressed in plant pollen

[0032] 1) Preparation of Arabidopsis thaliana genomic DNA

[0033] About 100 mg of young Arabidopsis thaliana leaves were placed in a 2 mL sterile centrifuge tube with 1 steel ball; quick-frozen in liquid nitrogen for about 5 min, and quickly ground into powder by a grinder at 35 Hz for 20 s; add 400 μL of preheated 65 ℃ The CTAB extract was fully shaken and then placed at 65°C and heated for 30 minutes (shaking every 10 minutes); the sample was taken out and cooled at room temperature, added with the same volume of chloroform (400 μL) as CTAB, and gently inverted up and down several times, and allowed to stand at room temperature for 5 minutes to make It is fully emulsified; centrifuge at 12,000 rpm for 1 min; take 200-400 μL of supernatant into a 1.5-mL sterile centrifuge tube, add 1 mL of pre-cooled absolute ethanol, and invert several times; ; Add 50 μL of sterile water, incuba...

Embodiment 2

[0041] Example 2: Construction of recombinant expression vector pCP09-GUS of promoter CP09

[0042] 1) With the primer design method of Example 1, obtain the PCR amplification primer pair of the promoter CP09, first add HindIII and BamHI restriction sites at the 5' ends of the forward primer and the reverse primer, respectively, and further respectively in the forward and reverse directions. A 15bp sequence overlapping the corresponding junction position of the pBI101 carrier was added to the 5' end of the primer to obtain a pair of PCR amplification primers for connecting to the promoter CP09 of the pBI101 carrier, the forward primer and reverse primer sequence are respectively as SEQ ID No.4 and SEQ ID No.5.

[0043] 2) utilize the above-obtained promoter CP09 amplification primer pair (sequence as shown in SEQ ID No.4 and SEQ ID No.5), carry out PCR amplification with pMD19-CP09 plasmid as a template, and carry out gel recovery to the PCR product, The target fragment was o...

Embodiment 3

[0046] Example 3: Transforming the recombinant expression vector pCP09-GUS into Arabidopsis thaliana

[0047] 1) Transfer the recombinant expression vector pCP09-GUS into Agrobacterium

[0048] Take 1 μg-2 μg of the recombinant expression vector pCP09-GUS plasmid obtained in Example 2, and transform it into Agrobacterium GV3101 competent cells by freeze-thaw method. ) on the sterile YEB solid plate to culture and screen single clones, use specific primers SEQ ID No.6 and SEQ ID No.7 to carry out PCR verification to pCP09-GUS recombinant bacteria, confirm positive clones, PCR reaction system and procedure are the same as the embodiment 1.

[0049] 2) Agrobacterium-mediated genetic transformation of Arabidopsis thaliana

[0050] The pCP09-GUS Agrobacterium strain was activated at 28°C, and the wild-type Arabidopsis thaliana was transformed by the inflorescence dip method. -100mL of sterile YEB liquid medium containing rifampicin (25μg / mL) and kanamycin (50μg / mL), shake overni...

technical field

[0001] The invention relates to the field of plant genetic engineering and molecular biology, in particular to a strong promoter CP09 specifically expressed in plant pollen and its application. Background technique

[0002] The regulation of plant gene expression is mainly at the transcriptional level, which is coordinated by a variety of cis-acting elements and trans-acting factors. Promoters are important cis-acting elements that play a key role in transcriptional regulation. Promoters can be divided into three categories according to different expression modes, namely constitutive promoters, inducible promoters and tissue-specific promoters. , tubers, vascular bundles, flowers, pollen and seeds, etc.) to drive gene expression, to achieve timed and quantitative precise regulation of exogenous gene expression in plants, which can effectively reduce the energy loss or energy loss caused by the continuous expression of constitutive promoters in different part...

Examples

Embodiment 1

[0031] Example 1: Obtainment of a strong promoter CP09 specifically expressed in plant pollen

[0032] 1) Preparation of Arabidopsis thaliana genomic DNA

[0033] About 100 mg of young Arabidopsis thaliana leaves were placed in a 2 mL sterile centrifuge tube with 1 steel ball; quick-frozen in liquid nitrogen for about 5 min, and quickly ground into powder by a grinder at 35 Hz for 20 s; add 400 μL of preheated 65 ℃ The CTAB extract was fully shaken and then placed at 65°C and heated for 30 minutes (shaking every 10 minutes); the sample was taken out and cooled at room temperature, added with the same volume of chloroform (400 μL) as CTAB, and gently inverted up and down several times, and allowed to stand at room temperature for 5 minutes to make It is fully emulsified; centrifuge at 12,000 rpm for 1 min; take 200-400 μL of supernatant into a 1.5-mL sterile centrifuge tube, add 1 mL of pre-cooled absolute ethanol, and invert several times; ; Add 50 μL of sterile water, incuba...

Embodiment 2

[0041] Example 2: Construction of recombinant expression vector pCP09-GUS of promoter CP09

[0042] 1) With the primer design method of Example 1, obtain the PCR amplification primer pair of the promoter CP09, first add HindIII and BamHI restriction sites at the 5' ends of the forward primer and the reverse primer, respectively, and further respectively in the forward and reverse directions. A 15bp sequence overlapping the corresponding junction position of the pBI101 carrier was added to the 5' end of the primer to obtain a pair of PCR amplification primers for connecting to the promoter CP09 of the pBI101 carrier, the forward primer and reverse primer sequence are respectively as SEQ ID No.4 and SEQ ID No.5.

[0043] 2) utilize the above-obtained promoter CP09 amplification primer pair (sequence as shown in SEQ ID No.4 and SEQ ID No.5), carry out PCR amplification with pMD19-CP09 plasmid as a template, and carry out gel recovery to the PCR product, The target fragment was o...

Embodiment 3

[0046] Example 3: Transforming the recombinant expression vector pCP09-GUS into Arabidopsis thaliana

[0047] 1) Transfer the recombinant expression vector pCP09-GUS into Agrobacterium

[0048] Take 1 μg-2 μg of the recombinant expression vector pCP09-GUS plasmid obtained in Example 2, and transform it into Agrobacterium GV3101 competent cells by freeze-thaw method. ) on the sterile YEB solid plate to culture and screen single clones, use specific primers SEQ ID No.6 and SEQ ID No.7 to carry out PCR verification to pCP09-GUS recombinant bacteria, confirm positive clones, PCR reaction system and procedure are the same as the embodiment 1.

[0049] 2) Agrobacterium-mediated genetic transformation of Arabidopsis thaliana

[0050] The pCP09-GUS Agrobacterium strain was activated at 28°C, and the wild-type Arabidopsis thaliana was transformed by the inflorescence dip method. -100mL of sterile YEB liquid medium containing rifampicin (25μg / mL) and kanamycin (50μg / mL), shake overni...

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