CN113185590B - Gene for regulating early heading and flowering of rice and application thereof - Google Patents

Abstract

The invention discloses a gene for regulating early heading and flowering of rice and its application. The amino acid sequence of the functional protein regulating early heading and flowering of rice is shown in SEQ ID NO.2. The present invention for the first time successfully cloned the gene OsGF14C gene that regulates the early heading and flowering stage of rice from the rice variety BC-10, and used the Camv35S promoter to construct a rice transformation vector that overexpresses the OsGF14C gene. The expression level of OsGF14C gene can be significantly increased after the vector is transformed into rice. Compared with the wild-type plants and other rice plants germinated, transplanted and managed at the same period, the flowering stage of the transgenic plants overexpressing OsGF14C is earlier. Therefore, the technology of overexpressing the OsGF14C gene of the present invention can be applied to genetic engineering breeding of rice, and can be applied to production practice to promote genetic breeding of rice with a short growth period, realize early maturity and high yield of rice, and ensure food security.

Description

A gene regulating early heading and flowering of rice and its use

Technical field:

The invention belongs to the field of rice, and specifically relates to a gene for regulating early heading and flowering of rice and its application.

Background technique:

Rice is the staple food of more than 50% of the world's population and one of the most important food crops for human beings. However, with the increase of population and the reduction of arable land caused by human activities, the problem of food security has become increasingly prominent.

Flowering period is one of the important agronomic traits affecting rice yield. Rice growth can generally be divided into three stages. The first stage is the vegetative growth period from bud to young panicle; the second stage is the reproductive growth period from young panicle to flowering; the third stage is the mature period from flowering to full maturity. The time of the second and third stages of different rice varieties is relatively constant, but the first stage of different rice varieties is significantly different, which largely determines the length of rice growth period. The length of the growth period of a variety determines the area and season suitable for planting the variety. The development and utilization of rice early-maturing genes will help promote the genetic breeding of rice with a short growth period, realize early-maturing and high-yielding rice, and directly affect rice yield.

Heading and flowering of higher plants is a complex biological process. Cloning genes that regulate flowering and conducting research on the mechanism of gene regulation during flower initiation has always been a research hotspot in plant developmental biology. According to statistics, so far, 25 genes regulating rice flowering have been cloned, but not including the genes of the GF14 family. Studies have shown that the GF14 gene family is a class of defense genes that widely exist in eukaryotes. They combine with a large number of phosphorylated proteins to initiate signal transmission, transcription and defense pathway genes, and play a role in the response of plants to the external environment. played an important role. There are a total of 8 GF14 genes in rice species, and there is no related report that GF14 genes can regulate the flowering period of rice.

Invention content:

The first object of the present invention is to provide a functional protein that regulates the early heading and flowering stage of rice.

We constructed transgenic plants overexpressing OsGF14C, planted transgenic plants overexpressing OsGF14C and wild-type controls at the same time, we found that the transgenic plants overexpressing OsGF14C had earlier heading and anthesis. OsGF14C is a new gene that regulates the early flowering period of rice, and has important potential application value in the genetic breeding of promoting short growth period of rice.

The amino acid sequence of the functional protein regulating early heading and flowering of rice is shown in SEQ ID NO.2.

The second object of the present invention is to provide a gene encoding the above-mentioned functional protein that regulates the early heading and flowering stage of rice.

Preferably, the nucleotide sequence of the gene regulating early heading and flowering of rice is shown in the 32nd-802nd base sequence of SEQ ID NO.1. It should be understood that, considering the degeneracy of codons, modifying the nucleotide sequence of the above-mentioned coding gene without changing the amino acid sequence also falls within the protection scope of the present invention.

The third object of the present invention is the application of the functional protein or gene regulating the early heading and flowering stage of rice in regulating the early heading and flowering stage of rice.

Preferably, the application is in genetic breeding of short growth period of rice.

Preferably, the rice growth period is shortened by overexpressing the above-mentioned gene regulating the early heading and flowering period of rice in rice.

For the first time, the present invention successfully clones the OsGF14C gene, which regulates the early heading and flowering stage of rice, from the rice variety BC-10, and uses the Camv35S promoter to construct a rice transformation vector overexpressing the OsGF14C gene. The expression level of OsGF14C gene can be significantly increased after the vector is transformed into rice. Compared with the wild-type plants and other rice plants germinated, transplanted and managed at the same period, the flowering stage of the transgenic plants overexpressing OsGF14C is earlier. Therefore, the technology of overexpressing the OsGF14C gene of the present invention can be applied to genetic engineering breeding of rice, and can be applied to production practice to promote genetic breeding of rice with a short growth period, realize early maturity and high yield of rice, and ensure food security.

Description of drawings:

Figure 1 is the carrier map of PHQSN;

Figure 2 is the detection of OsGF14C gene expression level in transgenic plants, where CK represents wild type Lijiang Xintuan Heigu, and OE-1, OE-2, OE-3 are transgenic plants of different lines.

Fig. 3 is Lijiang Xintuan Heigu (LTH) plants of wild type Lijiang Xintuan Heigu (LTH) plants germinated and transplanted at the same time, transgenic plants with OsGF14C overexpression and other rice plants. Under the same management mode, overexpression of OsGF14C regulates the early heading and flowering of rice. In the picture The white arrows at indicate the extracted tassels.

Detailed ways:

The following examples are to further illustrate the present invention, rather than limit the present invention.

Example 1:

1. Cloning of OsGF14C gene, construction of overexpression vector

Take the leaves of rice variety BC-10, use TriZol Reagent (Invitrogen Company, its article number is: 15596026) to extract the total RNA of the leaves, use agarose gel electrophoresis and ultraviolet spectrophotometer to detect the purity and amount of total RNA, take 1 μg of total RNA RNA was used to initiate the reverse transcription reaction, and the reverse transcriptase used was PrimeScript (TAKARA Company), and the steps of the reverse transcription reaction were referred to the instructions for use of the reverse transcriptase. Using the reverse transcription product as a template, primers F: CGGAATTC (EcoR1 restriction site) CGCCACCGAAGTAATCCCTT and R: GAAGATCT (BglII restriction site) CCAATAACATGCGGAGCCAT were used for PCR amplification, and the polymerase used in PCR was KOD FX (Toyobo Company). The reaction system was 50uL, and the PCR reaction system was prepared according to the instructions of KOD FX. The reaction conditions are: 94°C for 5 min; 35 cycles of 94°C for 30 sec, 60°C for 30 sec, and 68°C for 60 sec; 68°C for 10 min. A fragment of about 843bp was obtained by PCR amplification (the specific nucleotide sequence is shown in SEQ ID NO.1). After the fragment was recovered by agarose gel electrophoresis, EcoR1 and BglII were used to perform double enzyme digestion on the PCR fragment and the pHQSN carrier (the vector diagram is shown in Figure 1, and its nucleotide sequence is shown in SEQ ID NO.3), Recover target fragment and carrier fragment after enzyme digestion, and connect with T4 ligase (NEB company) at 16°C for 16 hours. The ligation system is: T4 ligase 1uL, 10Xbuffer 1uL, gene digestion fragment 6ul (200ng), pHQSN vector digestion Fragment 2 uL (50 ng). Take 1 uL of the ligation product and transform it into Escherichia coli DH5α by electric shock method, and coat the transformed product with kanamycin-resistant LB solid medium. Cultivate overnight at 37°C, pick 10 single clones to extract plasmids, and identify by enzyme digestion. Two positive clones were selected for sequencing detection. Sequencing found that the amplified fragment sequence had a total length of 843 bases, and its nucleotide sequence was shown in SEQ ID NO.1, which contained an open reading frame of 771 bases, namely It is the OsGF14C gene, its nucleotide sequence is shown in the 32nd-802nd base sequence of SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2. And obtain the overexpression vector pHQSN that carries the OsGF14C gene, named: overexpression vector pHQSN-OX, it is to insert the OsGF14C gene fragment into the Camv35S promoter downstream of the overexpression vector pHQSN between the restriction sites of EcoR1 and BglII .

2. Transformation of OsGF14C gene

The overexpression vector pHQSN-OX was transformed into Lijiang Xintuan Heigu by the method of genetic transformation mediated by Agrobacterium EHA105. The transformed primary generation (T0 generation) was detected by PCR and quantitative PCR (GF14CqF (ACTGCTGCAGAATCTAAGGTG) and GF14CqR (CCAGAGCAATATCCTGAGCA) primers (the product length is 145bp)), and the positive transformed plants were identified from the DNA level and RNA level. Positive plants were self-crossed to obtain transgenic 1st generation (T1 generation) strains. For each strain, 10 plants that were detected as positive by PCR were selected for propagation to obtain T2 generation plants. The T2 generation strains were then subjected to PCR detection to find out the origin of different There were 3 homozygous lines (OE-1, OE-2, OE-3) of the T2 generation plants of the T0 generation plants. Using the wild-type results as a control, the expression of the target gene OsGF14C in these three strains was detected by fluorescent quantitative PCR technology.

Fluorescent quantitative PCR to identify the overexpression effect of OsGF14C gene in transgenic plants The procedure is as follows:

Get the transgenic plants (OE-1, OE-2, OE-3) three-leaf stage seedling leaves of positive PCR detection to carry out the reagent that total RNA extraction adopts is TriZol Reagent (Invitrogen Company, its article number is: 15596026), according to the reagent The steps in the instructions were carried out, and the purity and amount of total RNA were detected by agarose gel electrophoresis and ultraviolet spectrophotometer, and 1 μg of total RNA was taken for initial reverse transcription reaction. The reverse transcriptase used was PrimeScript (Takara Company), reverse For the steps of the transcription reaction, refer to the instructions for use of the reverse transcriptase. Using the reverse transcription product as a template, the primers GF14CqF (ACTGCTGCAGAATCTAAGGTG) and GF14CqR (CCAGAGCAATATCCTGAGCA) primers were used to detect the expression of OsGF14C gene, and the rice EF1a gene was detected by EF1aqF (TTTCACTCTTGGTGTGAAGCAGAT) and EF1aqR (GACTTCCTTCACGATTTCATCGTAA) primers of rice housekeeping gene EF1a gene The expression was used as an internal reference, and the wild-type Lijiang Xintuan Heigu was used as a control. The test results are shown in Figure 2. The results showed that the expression of OsGF14C gene in the three transgenic plant lines was increased compared with the wild type.

3. Comparison of heading and flowering stages between OsGF14C gene overexpression transgenic plants and wild-type control

The seeds of the above-mentioned three homozygous overexpression transgenic T2 generation lines (OE-1, OE-2, OE-3) and the wild-type Lijiang Xintuan Heigu plant were placed at 49°C for 96h to break dormancy, and placed Soak in water for 36 hours after room temperature, germinate for 48 hours at 30°C, select seeds with the same germination state to sow in the same soil, and transplant seedlings uniformly after the 3-leaf stage. Observe the heading and flowering situation. The results are shown in Figure 3, compared with the wild-type LTH plants that were sown and transplanted at the same time, the heading and flowering stage of the transgenic rice plants overexpressing OsGF14C was earlier.

In summary, the gene OsGF14C of rice variety BC-10 is a functional gene that regulates the early heading and flowering stage of rice, and the transgenic plants overexpressing OsGF14C have early heading and flowering stage.

sequence listing

<110> Rice Research Institute of Guangdong Academy of Agricultural Sciences

<120> A gene regulating early heading and flowering in rice and its application

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atgtgtggag gaacgggcgg ttggccaggc gtaagcggct gggttgtctg ccggccctgc 4620

aatggcactg gaacccccaa gcccgaggaa tcggcgtgac ggtcgcaaac catccggccc 4680

ggtacaaatc ggcgcggcgc tgggtgatga cctggtggag aagttgaagg ccgcgcaggc 4740

cgcccagcgg caacgcatcg aggcagaagc acgccccggt gaatcgtggc aagcggccgc 4800

tgatcgaatc cgcaaagaat cccggcaacc gccggcagcc ggtgcgccgt cgattaggaa 4860

gccgcccaag ggcgacgagc aaccagattt tttcgttccg atgctctatg acgtgggcac 4920

ccgcgatagt cgcagcatca tggacgtggc cgttttccgt ctgtcgaagc gtgaccgacg 4980

agctggcgag gtgatccgct acgagcttcc agacgggcac gtagaggttt ccgcagggcc 5040

ggccggcatg gccagtgtgtggggattacga cctggtactg atggcggttt cccatctaac 5100

cgaatccatg aaccgatacc gggaagggaa gggagacaag cccggccgcg tgttccgtcc 5160

acacgttgcg gacgtactca agttctgccg gcgagccgat ggcggaaagc agaaagacga 5220

cctggtagaa acctgcattc ggttaaacac cacgcacgtt gccatgcagc gtacgaagaa 5280

ggccaagaac ggccgcctgg tgacggtatc cgagggtgaa gccttgatta gccgctacaa 5340

gatcgtaaag agcgaaaccg ggcggccgga gtacatcgag atcgagctag ctgattggat 5400

gtaccgcgag atcacagaag gcaagaaccc ggacgtgctg acggttcacc ccgattactt 5460

tttgatcgat cccggcatcg gccgttttct ctaccgcctg gcacgccgcg ccgcaggcaa 5520

ggcagaagcc agatggttgt tcaagacgat ctacgaacgc agtggcagcg ccggagagtt 5580

caagaagttc tgtttcaccg tgcgcaagct gatcgggtca aatgacctgc cggagtacga 5640

tttgaaggag gaggcggggc aggctggccc gatcctagtc atgcgctacc gcaacctgat 5700

cgagggcgaa gcatccgccg gttcctaatg tacggagcag atgctagggc aaattgccct 5760

agcaggggaa aaaggtcgaa aaggtctctt tcctgtggat agcacgtaca ttgggaaccc 5820

aaagccgtac attgggaacc ggaacccgta cattgggaac ccaaagccgt aattgggaa 5880

ccggtcacac atgtaagtga ctgatataaa agagaaaaaa ggcgattttt ccgcctaaaa 5940

ctctttaaaa ctctttaaaa ctcttaaaac ccgcctggcc tgtgcataac tgtctggcca 6000

gcgcacagcc gaagagctgc aaaaagcgcc tacccttcgg tcgctgcgct ccctacgccc 6060

cgccgcttcg cgtcggccta tcgcggccgc tggccgctca aaaatggctg gcctacggcc 6120

aggcaatcta ccagggcgcg gacaagccgc gccgtcgcca ctcgaccgcc ggcgcccaca 6180

tcaaggcacc ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 6240

tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 6300

gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata 6360

gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca 6420

tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc 6480

cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 6540

tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 6600

gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 6660

ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 6720

aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 6780

tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 6840

ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 6900

gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 6960

tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 7020

caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 7080

ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 7140

cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 7200

ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 7260

cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 7320

gcattctagg tactaaaaca attcatccag taaaatataa tattttattt tctcccaatc 7380

aggcttgatc cccagtaagt caaaaaatag ctcgacatac tgttcttccc cgatatcctc 7440

cctgatcgac cggacgcaga aggcaatgtc atacacttg tccgccctgc cgcttctccc 7500

aagatcaata aagccactta ctttgccatc tttcacaaag atgttgctgt ctcccaggtc 7560

gccgtgggaa aagacaagtt cctcttcggg cttttccgtc tttaaaaaat catacagctc 7620

gcgcggatct ttaaatggag tgtcttcttc ccagttttcg caatccacat cggccagatc 7680

gttattcagt aagtaatcca attcggctaa gcggctgtct aagctattcg tatagggaca 7740

atccgatatg tcgatggagt gaaagagcct gatgcactcc gcatacagct cgataatctt 7800

ttcagggctt tgttcatctt catactcttc cgagcaaagg acgccatcgg cctcactcat 7860

gagcagattg ctccagccat catgccgttc aaagtgcagg acctttggaa caggcagctt 7920

tccttccagc catagcatca tgtccttttc ccgttccaca tcataggtgg tccctttata 7980

ccggctgtcc gtcattttta aatataggtt ttcattttct cccaccagct tatatacctt 8040

agcaggagac attccttccg tatcttttac gcagcggtat ttttcgatca gttttttcaa 8100

ttccggtgat attctcattt tagccatta ttaatttcctt cctcttttct acagtattta 8160

aagatacccc aagaagctaa ttataacaag acgaactcca attcactgtt ccttgcattc 8220

taaaacctta aataccagaa aacagctttt tcaaagttgt tttcaaagtt ggcgtataac 8280

atagtatcga cggagccgat tttgaaaccg cggtgatcac aggcagcaac gctctgtcat 8340

cgttacaatc aacatgctac cctccgcgag atcatccgtg tttcaaaccc ggcagcttag 8400

ttgccgttct tccgaatagc atcggtaaca tgagcaaagt ctgccgcctt acaacggctc 8460

tcccgctgac gccgtcccgg actgatgggc tgcctgtatc gagtggtgat tttgtgccga 8520

gctgccggtc ggggagctgt tggctggctg gtggcaggat atattgtggt gtaaacaaat 8580

tgacgcttag acaacttaat aacacattgc ggacgttttt aatgtactga attaacgccg 8640

aattaattcg ggggatctgg atttagtac tggattttgg ttttaggaat tagaaatttt 8700

attgatagaa gtattttaca aatacaaata catactaagg gtttcttata tgctcaacac 8760

atgagcgaaa ccctataggga accctaattc ccttatctgg gaactactca cacatttatta 8820

tggagaaact cgagcttgtc gatcgacaga tccggtcggc atctactcta tttctttgcc 8880

ctcggacgag tgctggggcg tcggtttcca ctatcggcga gtacttctac acagccatcg 8940

gtccagacgg ccgcgcttct gcgggcgatt tgtgtacgcc cgacagtccc ggctccggat 9000

cggacgattg cgtcgcatcg accctgcgcc caagctgcat catcgaaatt gccgtcaacc 9060

aagctctgat agagttggtc aagaccaatg cggagcatat acgcccggag tcgtggcgat 9120

cctgcaagct ccggatgcct ccgctcgaag tagcgcgtct gctgctccat acaagccaac 9180

cacggcctcc agaagaagat gttggcgacc tcgtattggg aatccccgaa catcgcctcg 9240

ctccagtcaa tgaccgctgt tatgcggcca ttgtccgtca ggacattgtt ggagccgaaa 9300

tccgcgtgca cgaggtgccg gacttcgggg cagtcctcgg cccaaagcat cagctcatcg 9360

agagcctgcg cgacggacgc actgacggtg tcgtccatca cagtttgcca gtgatacaca 9420

tggggatcag caatcgcgca tatgaaatca cgccatgtag tgtattgacc gattccttgc 9480

ggtccgaatg ggccgaaccc gctcgtctgg ctaagatcgg ccgcagcgat cgcatccata 9540

gcctccgcga ccggttgtag aacagcgggc agttcggttt caggcaggtc ttgcaacgtg 9600

acaccctgtg cacggcggga gatgcaatag gtcaggctct cgctaaactc cccaatgtca 9660

agcacttccg gaatcggggag cgcggccgat gcaaagtgcc gataaacata acgatctttg 9720

tagaaaccat cggcgcagct atttacccgc aggacatatc cacgccctcc tacatcgaag 9780

ctgaaagcac gagattcttc gccctccgag agctgcatca ggtcggagac gctgtcgaac 9840

ttttcgatca gaaacttctc gacagacgtc gcggtgagtt caggcttttt catatctcat 9900

tgccccccgg gatctgcgaa agctcgag 9928

Claims (2)

1. The application of the functional protein regulating the early heading and flowering stage of rice or its coding gene in regulating the early heading and flowering stage of rice, the amino acid sequence of the functional protein regulating the early heading and flowering stage of rice is as shown in SEQ ID NO.2 , the application is to overexpress in rice the gene that regulates the early heading and flowering stage of rice so as to shorten the growth period of rice. 2. application according to claim 1, is characterized in that, the nucleotide sequence of the gene of the regulating paddy rice heading and flowering period of coding described functional protein such as the 32nd base of SEQ ID NO.1-the 802nd The base sequence is shown.

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