CN105624157A - Drought-induced pollen specific promoter and use thereof - Google Patents

Abstract

The invention belongs to the technical fields of molecular biology and genetic engineering, and specifically relates to a rice pollen-specific promoter and application thereof. In the present invention, the promoter sequence of rice <i>OsCLO7</i> gene is shown in SEQ ID NO.1. The promoter activity was affected by anther development and drought stress. Quantitative PCR results showed that: <i>OsCLO7</i> was mainly expressed in rice panicle, more specifically in the stamen of panicle tissue; the expression increased with the progress of development; the expression was induced by drought stress in the early stage of young panicle development . The GUS reporter gene driven by this promoter was expressed in rice anthers at different developmental stages after meiosis, more specifically in the pollen grains of anthers; GUS activity was induced by drought in the early stage of panicle development and was limited to pollen grains. The promoter in the present invention can be used to construct a transgene carrier that drives the specific expression of foreign genes in pollen grains, and has important application value in agricultural production.

Description

A drought-induced pollen-specific promoter and its application

technical field

The invention belongs to the technical fields of molecular biology and genetic engineering, and in particular relates to a drought-induced pollen-specific promoter and application thereof.

Background technique

The promoter is a part of the gene, usually located in the upstream region of the start codon at the 5 ^' end. This region contains transcriptional regulatory factors, DNA sequences that RNA polymerase specifically recognizes and binds to, and controls the initiation time and degree of gene expression (transcription). Promoters are like "switches" that determine the activity of genes. The promoters of plant genes are divided into constitutive promoters, inducible promoters and tissue-specific promoters according to their gene expression methods.

Constitutive promoters refer to promoters that can efficiently promote the expression of exogenous genes in all or most of the plant tissues, while tissue-specific promoters drive genes that are only specific in one or some processes of plant growth. expressed in organs or tissues. For example, leaf-specific promoters, stamen-specific promoters, and the like. Discovering these promoters and identifying their activities will not only help elucidate the role of genes in plant growth and development, but also drive the effective expression of target genes in specific tissues with the help of these promoters, which can be applied in scientific research and agriculture Production Practice.

An inducible promoter means that when certain conditions such as physical, chemical, and biological signals exist, the promoter can drive the enhanced expression of downstream genes, and the transcription level is greatly increased. Due to long-term exposure to the natural environment, plants are always affected by environmental stress. Correspondingly, the expression of a large number of genes in the evolution process is induced by environmental stress factors, such as high temperature, drought, and pests, and regulates signaling pathways and molecular activities in the body. , against adversity. With the intensification of the greenhouse effect and the sharp increase of industrial and agricultural water use, drought has become the main factor threatening agricultural production. The reproductive growth stage of crops, especially the development of stamens, is most susceptible to environmental stress, resulting in the most severe yield reduction. Therefore, discovering the drought stress-responsive genes specific to stamen development and using their promoters to drive the expression of target genes is of great significance for the study of plant reproduction and drought resistance and plant genetic engineering.

The OsCLO 7 gene involved in the present invention is one of the eight members of the rice caleosins (CLOs) gene family. The expression of the gene in pollen grains increases with the development of young spikes and is induced by drought stress. The invention provides the driving activity of the OsCLO7 gene promoter, the promoter can drive the specific expression of the gene in rice pollen grains, and its activity is enhanced under drought stress, and it can be predicted that it has strong application value in scientific research or production practice .

Contents of the invention

The purpose of the present invention is to provide a tissue-specific promoter and its application, especially to provide an inducible promoter induced by drought stress and its application.

The invention includes the cloning of the promoter sequence, the construction of the plant transgenic vector, the activity analysis of the promoter driving gene expression in different tissues, and the analysis of the response of the promoter to drought stress. The promoter belongs to a tissue-specific promoter and responds to drought stress, so it is of great value in the field of research on rice reproduction and drought resistance and in the field of plant transgenic technology.

The promoter provided by the present invention is a DNA sequence with a size of about 2224bp cloned from the upstream of the initiation codon of rice OsCLO 7 gene (Os06g0254600), and its nucleotide sequence is shown in SEQ ID NO.1.

The present invention also includes a genetic engineering vector constructed using the promoter (SEQ ID NO.1 sequence);

The present invention also includes the application of the promoter (SEQ ID NO.1 sequence) as a pollen grain-specific promoter in genetic engineering, and the promoter can drive the specific expression of the target gene in the pollen of the 9-12 stage of stamen development .

The present invention also includes the use of the promoter (SEQ ID NO.1 sequence) as a drought-inducible promoter in anthers in the early stage of stamen development, and the promoter can drive the expression of a target gene in developing pollen grains under drought stress.

The promoter in the present invention can be used to construct a transgenic carrier that drives the specific expression of foreign genes in pollen grains, and has important application value in both scientific research and agricultural production.

The acquisition of the promoter is derived from the research on the expression characteristics of the OsCLO7 gene and the research on the drought resistance of the gene. First, the expression characteristics of this gene were obtained from the drought stress response microarray data during rice panicle development. OsCLO7 is the only gene among the eight rice calpains that is both developmentally regulated and drought stress-induced (see Figure 1 ); Quantitative PCR was used to detect the expression of this gene, and the expression profile of each tissue was analyzed, and it was found that it was only specifically expressed in stamens (see Figure 2). Comparing the expression of OsCLO7 in various tissues after drought, it was found that its expression was induced by drought in the early stage of young panicle development, especially during the meiosis period, and the background expression was already very high after meiosis, and it was no longer induced by drought stress (see Figure 3 ).

The promoter was constructed in a vector containing the reporter gene GUS ( β-glucuronidase , encoding glucuronidase) (see Figure 4), and transgenic positive strains were obtained through Agrobacterium-mediated rice transgenic technology. The GUS activity was detected in various rice tissues, and it was found that the GUS reporter gene driven by it was only highly expressed in the anthers at the later stage of rice panicle development, and more specifically expressed in pollen grains (see Figure 5). Therefore, the OsCLO7 gene promoter can drive the expression of foreign genes in specific tissues, so as to achieve the purpose of directional transgene.

The transgenic plants were subjected to various periods of drought treatment, and the GUS activity was detected, and the results were consistent with expectations, only in the anthers, and the expression was stronger in the anthers after drought. The GUS activity of anthers was hardly detected in the control materials at the early stage of panicle development, but the GUS activity of anthers was stronger in the materials at the corresponding period of drought, and the control and treated anthers at the pollen grain maturity stage had strong GUS activities (see Figure 6) .

Description of drawings

Figure 1 Microarray data analysis of the expression of rice calpain gene under drought stress in young panicles. There are 8 members in the rice calpain gene family, OsCLO1 to OsCLO7 . 2~3mm, 3~4mm, 4~5mm, 5~7mm are the length of florets. C is the control, D is the drought treatment. OsCLO7 was the only gene in the CLO family whose expression was significantly up-regulated in flowers at different stages after drought stress.

Figure 2 Tissue expression profile of OsCLO7 .

Figure 3 The drought-response expression profile of OsCLO7 .

Figure 4 Schematic diagram of the vector construction of the OsCLO7 gene promoter.

Fig. 5 Analysis of GUS activity in rice tissues. Among them, a is the rice seedling of one week (bar=1cm); b-d are the roots, stems and leaves of mature stage (bar=1mm); e, f, g, h, i are the 6th (bar=0.5mm), 8th, Spikelets at stages 10, 12, and 14 (bar=2mm); j, k, and l are pistils, anthers, and pollen grains at stages 12 (bar=0.1mm); m is an immature embryo pollinated for 4 days (bar=1mm ). GUS activity mainly appeared in late pollen grains.

Fig. 6 Comparison of GUS activity in anthers after drought treatment during panicle development (bar=0.1mm). After drought stress, the GUS signal appeared in the early stage of pollen grain development, and the signal intensity increased.

detailed description

The present invention is further illustrated below in conjunction with specific examples. It should be understood that these examples are only for illustrating the present invention and do not limit the scope of the present invention. The experimental methods without specific conditions indicated in the following examples are usually in accordance with conventional conditions, that is, the conditions described in the "Molecular Cloning" experimental manual by Sambrook et al., or in accordance with the conditions suggested by the manufacturer.

Embodiment 1 rice cultivation and drought treatment

The rice Nipponbare is used as the material, and it is planted in artificially controlled greenhouses and pots in outdoor natural environments. The drought treatment was carried out in the greenhouse. Drought treatment method: grow rice under normal conditions until the reproductive development period, then stop watering during the reproductive development period, and keep it for a week after the relative soil moisture content drops to 15-20%, and take samples for gene expression analysis and GUS staining .

Example 2 Gene chip analysis of gene expression in the rice panicle development process under drought conditions

After the rice Nipponbare seeds germinated, they were transplanted to the soil in the plastic pots of the greenhouse to grow. After 50 days, the growth status of the rice was closely observed and checked, and the drought treatment was carried out when the vegetative period was just transferred to the reproductive growth: pour out the excess water in the pot, stop Watering, when the soil moisture drops to about 30%, add a small amount of water every day to maintain the moisture content for a week, take rice flowers of different sizes (2~3mm, 3~4mm, 4~5mm, 5~7mm) and collect them separately And numbered, with rice under normal growth conditions as a control, do three repetitions. Chip analysis was done by Agilent.

Embodiment 3qRT-PCR detection

Rice samples from different tissues and different panicle development stages were taken, RNA was extracted according to the reagents and methods provided by the RNA extraction kit (RNAisoplus, TaKaRa), and reverse transcription was performed (PrimeScriptRTregentKitWithgDNAEraser, TaKaRa). In this example, the probe-based quantitative PCR method is adopted because the members of the OsCLO family have high homology. Designing Taqman-specific probes for different genes can better distinguish the homologous genes of the OsCLO family. The probe and primer sequences are shown in SEQ ID NO.2-SEQ ID NO.4:

Probe sequence: FAM-ACCTTGCCGCCAGTGT-MGB (SEQ ID NO.2);

Upstream primer: GTTGTGACTTCGCATTTGCTAGA (SEQ ID NO.3);

Downstream primer: CGATAAGTGAGGTAATGGTGCATC (SEQ ID NO. 4).

PCR reaction system:

2XPremixExTaq5μl

Primer-F0.2μl

Primer-R 0.2μl

Probe0.4μl

RT reaction solution 2μl

DyeI0.2μl

ddH2O4μl

PCR reaction program:

(1) Pre-denaturation at 95°C for 30s;

(2) Denaturation at 95°C for 5s, annealing at 60°C for 30s, cycle 40 times;

Example 4 Transgenic vector construction

The rice genomic DNA was extracted, and the OsCLO7 promoter was amplified by PCR technology. The primers used were the underlined part of the sequence shown in SEQ ID NO.1, namely: upstream primer: CTTCAGTTGTCTTTGCCTCC (SEQ ID NO.5), downstream primer: AGAGCAATTCTGCGAGACG (SEQ ID NO.6). The upstream primer introduces the Sal I restriction site, and the downstream introduces the Sma I restriction site. The amplified product was connected into the PCR-Blunt intermediate vector, and after the sequencing was correct, it was connected into the modified transgenic final vector pCAMBIA1300, transformed into Agrobacterium EHA105, and the presence of the promoter fragment in the positive clone was confirmed by colony PCR.

Embodiment 5 rice genetic transformation

A. Induction of callus

Select mature and plump seeds, remove the glume, soak in 70% ethanol for 2 minutes, then soak in sodium hypochlorite solution containing 2% available chlorine for 30 minutes, wash with sterilized water for 7-8 times, dry on absorbent paper, and place in N6D for cultivation Base, 28 ℃, 16h light culture for about 30 days, take the scattered callus subculture for 10 days.

B． Cultivation of Agrobacterium EHA105

Spread the Agrobacterium liquid containing the correct plasmid obtained in the previous part of the experiment on the YEB three-antibody plate (streptomycin, rifampicin and kanamycin resistance), culture in the dark at 28°C for about 36 hours, and collect the colonies in In the liquid co-culture medium, adjust its OD to about 0.2.

C． Infection and co-cultivation

Collect the subcultured callus in the prepared co-culture solution and let it stand for 30 minutes. Pour off the bacterial liquid, blot dry the callus on absorbent paper, connect it to the co-cultivation medium, and culture it in the dark at 20°C for 2-3 days.

D． filter

The callus was transferred to the selection medium (N6D+50mg/L hygromycin B+250mg/L cephalosporin) and light cultured at 28°C. The medium can be replaced every 15-20 days, and the screening time should not be less than 45 days.

E． differentiation

Transfer the newly grown callus through selection to differentiation medium (MS+2mg/L6-BA+0.2mg/LNAA+2mg/LKT+0.2mg/LIAA+50mg/L hygromycin+250mg/L cephalosporin ), 16 hours light culture at 28°C. Before the differentiation culture, the callus can be cultured in the dark for several days as pre-differentiation. Change medium regularly.

F． root

The differentiated transgenic seedlings (>1cm high) were stripped of excess callus, and the roots were cut off (leaving about 0.5cm), and moved to 1/2MS medium to take root. Incubate in light at 28°C for 16 hours.

G． Hardening and transplanting

After the rooting is finished, the rooting medium can be removed, and the seedlings are soaked in water for several days to harden the seedlings, and then transplanted into the soil to grow.

Example 6 GUS tissue staining

Take different tissues of T1 generation positive transgenic rice and soak in GUS staining solution [0.2mol/L Na ₂ HPO ₄ /NaH ₂ PO ₄ , 0.1mol/L K ₃ Fe(CN) ₆ /K ₄ Fe(CN) ₆ , 0.1% X-Gluc, 0.1% TritonX-100], stained at 37°C for 6 hours after aspirating, decolorized with absolute ethanol for two days, changed the medium several times during the period, and finally soaked in 70% ethanol for storage. Observed and photographed under a stereomicroscope (Leica S8APO).

Among them, SEQ ID NO.1 description:

(i) Sequence features: 2224bp long, linear nucleotides

(ii) Molecule type: Nucleic acid

(iii) Sequence and sequence markers: the underlines are the sequences of upstream and downstream primers

(iv) Blackened ATG is the start codon of OsCLO7 gene

1 CTTCAGTTGTCTTTGCCTCC TGTCTTGAGGTCCTGTTGAGATCCCATGGC50

51AAAATTTTTTACTATGTCACATCGAATGTTTGACACATGCATGAAGTATT100

101AAATATAAACGAAAAAAAAAACTAATTACACAGATTGCGTGTAAATTGCGA150

151GATGAATATTAAGTCTAATTGCTTCATGATCTGACAATGTAGTGCTATA200

201GTAAATATTTGCTAATGACGGATTAATTAGACTTAATAAATTCGTCTCGC250

251AGTTTATAGGCGGATTCTGTAATTTTATTTTGTTATTAGTCTATGTTTAAT300

301ACTTCAAATATGTGTCCGTATATCCGATGTGACACGCTAAAATTTTACAC350

351CCATAATCTAATTAAACACATCCTAAATCTAGTGAAGATATATACATACC400

401CGGCAGCTATATATAGTTGAAAGAGAGGTCATCACACATAATATATA450

451AAGTCCAAGACATGGTTTTTCATTGCATGCATGATGCATAGACAGCATA500

501GTTCAATATATGTGAAATGCATATGGTGAAAAAAAAATTGCCAGTAAAGA550

551TGCACGGTATATCCTATTGATATCTACTAGTAGTTTTTAGAACCACTCGG600

601TGATATATGTGAGTTGCTAAAAAGTTCAGCAATATATGGGCACTATATTT650

651AAAACCTAACGGGTGGACTTAATTATATACACTCATGACTTCACCACCAC700

701ACCACATGTCCACACCACATGTGATTTCCCTACTTCGGGAATATTTTATCA750

751GCTTGTATAACGACACTTATTGTTCCAAAAAAATACACTAATTAATATCC800

801ACTAAACAAAAAGAAATGAAAATTACACATTGAGTCTTCTTCAACTTTGTA850

851TGGTTTAGTTTCTGCCAAGCTTGCAGAAGAATCGTCATAGAGTTTTTAGA900

901CATGCGCATATTTGGCTCAACATATTCTGTATTTCCATTTCGTAATCCAA950

951TATATTCTCTTTATGAGTAGATCTAGAAATGCCAATTTACTTTTTCCATGA1000

1001TGTCATTATCTAAAAAACACATTTCCATGATGTAACAAGTTCTGCATAAA1050

1051TGAAGATTAGATTTTACTAAGGCCGAGTTGGGAGCACATTTTCAGCGCAC1100

1101GTAAAATAGAGTGGTCCATTAGCGCGTGATTAATTAAGCATTAGCTATTT1150

1151TTTTTAAAAAAAATGGATCAATATGATTTTTTAAGCAACTTTCGTATAGAT1200

1201TTGTAAAAAAAACGCACCGTTTAACGATTTAAAAAGCATACATGGAACACG1250

1251AGGAAGATGAGTTGGGAAAGTTAGTGAAATAACTCATAGCCATGTATTAC1300

1301ATATCAATCATAAATATATAAATATCTTGTTCATCAATGTATCATCGTAC1350

1351CTACCACCGAGAGGACCTCCTCCTCATTGTTAGGCCTTGTTTTGTTGGAG1400

1401GATATTTTTTGGATTTAGTTGTCACATCGTATATACGGACACATATTTAAA1450

1451GTATTAAATATAGTCTAATAACAAAATAAATTACAGATTCCGCCAGGAAA1500

1501TTACGAGACGAATTTATCAAGCCTAATTAATCCGTCGCTAGCAATTGTTT1550

1551ACTGTAGCACCACATTGTCAAATCATGGCATAATTAGATTTAAAAGATTT1600

1601ATCTTGTAATTTATACGTAATCTGTGTAATTGTTTTTTTCCTACATTTAAT1650

1651ACTCCATGCATGTGTCTAACAGGGCCTTAGTTCAATCGGATCCTGTTGGG1700

1701TGGCTATGGCAACTGCAACGCGTTAGCCTCCGTTAAATCAGATCTTGCAC1750

1751AACTGCTGTTCATGAGAGTACGATTCAAAATGAATACTACTTCCTCCGTT1800

1801TTACAATGTAAGCCATTCTAACATTTCCTATATTCACATTGATGTTAATG1850

1851AATCTAGATAGATATATATGTATATATTAATTAACATCAATATGAATATG1900

1901AAAAATGCTAGAATGACTTACATTGTGAAACGGAGGGAGTATAACACATG1950

1951TTAGAGCCGTTACTATCTATTTACATGTGGCTCAAATCTGACCACCTACT2000

2001TGCTCGATCCAACACTGTACTACCCATCATAACATCCGACTGCTTAACGG2050

2051GCGGTAGCTATAGCAATCGCACCCGATCCAGAGTCTTGTTAGTTCTTTTT2100

2101TTTTTGAAACCTAAAAGTCTTGTTAGTTCTTTTTTTTTGAAACATAAAAG2150

2151TCTTGTTAGTTTCTTCCCTAGCTAGCTATTTAAGCTGGTATATGCGATTCC2200

2201ATTTG AGAGCAATTCTGCGAGACG atg 2227.

SEQUENCELISTING

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Claims (4)

1. pollen specific promoter one kind drought-induced, it is characterised in that from Oryza sativa L.OsCLOThe DNA sequence of the about 2224bp size that 7 gene start codon upstreams are cloned into, its nucleotides sequence is classified as shown in SEQIDNO.1.

2. the engineering carrier that the promoter utilized described in claim 1 builds.

3. promoter as claimed in claim 1 is as the application in genetic engineering of the pollen grain specific promoter, and this promoter can drive genes of interest specific expressed in the pollen of stamen development 9-12 phase.

4. promoter as claimed in claim 1 is as the application of drought-inducible promoter in stamen development early stage flower pesticide, and this promoter can drive destination gene expression in developmental pollen grain under drought stress.