IPC분류정보
국가/구분 |
United States(US) Patent
공개
|
국제특허분류(IPC7판) |
|
출원번호 |
17617601
(2020-10-19)
|
공개번호 |
20220307041
(2022-09-29)
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우선권정보 |
CN-202010631475.6 (2020-07-03) |
국제출원번호 |
PCT/CN2020/118264
(2020-10-19)
|
발명자
/ 주소 |
- ZHANG, Xiaojun
- LI, Jihua
- GUO, Rui
- YU, Xiaona
- SI, Tong
- ZOU, Xiaoxia
- WANG, Yuefu
- WANG, Minglun
- CHI, Xiaoyuan
- YU, Shanlin
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출원인 / 주소 |
- Qingdao Agricultural University
|
인용정보 |
피인용 횟수 :
0 인용 특허 :
0 |
초록
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Cloning and use of an Arachis hypogaea L. flowering habit gene AhFH1 and allelic variants thereof are provided. Through experiments, the Arachis hypogaea L. flowering habit gene AhFH1 and two defunctionalized allelic variants thereof are determined. The defunctionalized allelic variants can cause th
Cloning and use of an Arachis hypogaea L. flowering habit gene AhFH1 and allelic variants thereof are provided. Through experiments, the Arachis hypogaea L. flowering habit gene AhFH1 and two defunctionalized allelic variants thereof are determined. The defunctionalized allelic variants can cause the change from alternate flowering Arachis hypogaea L. to continuous flowering Arachis hypogaea L. Through overexpression of the gene AhFH1 or supplementary expression of a promoter of the gene itself, a continuous flowering Arachis hypogaea L. variety can be changed into alternate flowering Arachis hypogaea L.; and through knockout or expression suppression of the gene AhFH1, alternate flowering Arachis hypogaea L. can be changed into continuous flowering Arachis hypogaea L. Marker-assisted selection (MAS) breeding is realized for allelic variants of the gene using molecular markers.
대표청구항
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1. A method of using an Arachis hypogaea L. flowering habit gene AhFH1 and allelic variants thereof in crop genetic improvement, wherein the method is configured to improve a flowering habit and traits related to the flowering habit, the traits comprise shoot number, pod number, pod concentration, m
1. A method of using an Arachis hypogaea L. flowering habit gene AhFH1 and allelic variants thereof in crop genetic improvement, wherein the method is configured to improve a flowering habit and traits related to the flowering habit, the traits comprise shoot number, pod number, pod concentration, maturity consistency, and pod yield; the Arachis hypogaea L. flowering habit gene AhFH1 has a nucleotide sequence shown in SEQ ID NO: 1, cDNA encoded by the Arachis hypogaea L. flowering habit gene has a nucleotide sequence shown in SEQ ID NO: 2, and a protein encoded by the Arachis hypogaea L. flowering habit gene has an amino acid sequence shown in SEQ ID NO: 3; there are mainly two promoter sequences shown in SEQ ID NOs: 13-14 for the Arachis hypogaea L. flowering habit gene AhFH1, and the two promoter sequences are from Tifrunner and shitouqi, respectively; allelic variants of the Arachis hypogaea L. flowering habit gene AhFH1 comprise a defunctionalized allelic variant Ahfh1-1 and a defunctionalized allelic variant Ahfh1-2; the defunctionalized allelic variant Ahfh1-1 has a nucleotide sequence shown in SEQ ID NO: 8, has a 1,492 bp deletion from +1,872 bp to +3,273 bp at a genome-wide gene termini involving the last exon and 3′UTR and starting from ATG, and corresponds to a continuous flowering habit of Arachis hypogaea L.; and the defunctionalized allelic variant Ahfh1-2 has a nucleotide sequence shown in SEQ ID NO:11, and cDNA encoded by the defunctionalized allelic variant Ahfh1-2 has a sequence shown in SEQ ID NO: 12 and has a base C deletion at +335 bp, causing a cDNA translation frame frameshift to form a terminator in advance and thus making a translated protein incomplete, the defunctionalized allelic variant Ahfh1-2 corresponds to the continuous flowering habit of Arachis hypogaea L. 2. Cloning primers for an Arachis hypogaea L. flowering habit gene AhFH1, specifically comprising: a cloning primer pair FH1g-F/R for the Arachis hypogaea L. flowering habit gene AhFH1 at a genomic level, nucleotide sequences of the cloning primer pair FH1g-F/R are shown in SEQ ID NO: 4 and SEQ ID NO: 5; a cloning primer pair FH1cd-F/R for the Arachis hypogaea L. flowering habit gene AhFH1 at a cDNA level, nucleotide sequences of the cloning primer pair FH1cd-F/R are shown in SEQ ID NO: 6 and SEQ ID NO: 7; and a cloning primer pair FH1p-F/R for two different promoters of the Arachis hypogaea L. flowering habit gene AhFH1, nucleotide sequences of the cloning primer pair FH1p-F/R are shown in SEQ ID NO: 15 and SEQ ID NO: 16, the cloning primer pair FH1p-F/R is configured to identify the two different promoters of the Arachis hypogaea L. flowering habit gene AhFH1. 3. A functional molecular marker InDel-1492 bp for distinguishing a fully-functional Arachis hypogaea L. flowering habit gene AhFH1 and a defunctionalized Arachis hypogaea L. flowering habit gene Ahfh1-1, wherein a corresponding primer pair InDel-1492 bp-F/R has nucleotide sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10; and when it is confirmed in combination with sequencing that there is no mutation in an AhFH1 sequence, the functional molecular marker InDel-1492 bp is configured to directly distinguish the fully-functional Arachis hypogaea L. flowering habit gene AhFH1 and the defunctionalized Arachis hypogaea L. flowering habit gene Ahfh1-1 through PCR and electrophoresis. 4. A construction method and use of an overexpression transgenic vector carrying an Arachis hypogaea L. flowering habit gene AhFH1, wherein a 35S promoter of tobacco mosaic virus (TMV) is configured to construct the overexpression transgenic vector p35S::AhFH1 carrying a nucleotide sequence related to the Arachis hypogaea L. flowering habit gene AhFH1, with a plant overexpression vector PHB as a vector backbone; the construction method of the overexpression vector requires a primer pair of OE-FH1-F and OE-FH1-R, with sequences shown in SEQ ID NOs: 17-18; the primer pair is configured to amplify in cDNA of alternate flowering Arachis hypogaea L. or a plasmid carrying a complete coding frame of the Arachis hypogaea L. flowering habit gene to obtain the Arachis hypogaea L. flowering habit gene AhFH1, and an amplification product is introduced into the plant overexpression vector PHB or another plant overexpression vector through enzyme digestion or recombination to construct the overexpression transgenic vector p35S::AhFH1; and the overexpression vector is transformed into continuous flowering Arachis hypogaea L. to increase a shoot number of the continuous flowering Arachis hypogaea L., thereby affecting other related traits. 5. A construction method and use of a complementary expression transgenic vector carrying a nucleotide sequence related to an Arachis hypogaea L. flowering habit gene AhFH1, wherein on the basis of the an overexpression transgenic vector p35S::AhFH1, a promoter of the Arachis hypogaea L. flowering habit gene AhFH1 itself is configured to construct the complementary expression transgenic vector pFH1::AhFH1 carrying the nucleotide sequence related to the Arachis hypogaea L. flowering habit gene AhFH1; the construction method of the complementary expression vector requires a primer pair of FH1pro-F/R, with sequences shown in SEQ ID NOs: 19-20, wherein an upstream primer FH1pro-F has an EcoR I restriction site of “gaattc”, and a downstream primer FH1pro-R has a Pst I restriction site of “ctgcag”; the primer pair is used configured to clone and amplify a promoter of DNA of an alternate flowering Arachis hypogaea L. variety, an amplification product or a T vector carrying the amplification product is directly digested with EcoR I and Pst I, and then a target fragment is recovered and ligated with a large fragment recovered after the overexpression transgenic vector p355::AhFH1 undergoes the same digestion linearization to construct the complementary expression transgenic vector pFH1::AhFH1; and the complementary expression transgenic vector pFH1::AhFH1 is transformed into continuous flowering Arachis hypogaea L. to change the continuous flowering Arachis hypogaea L. into alternate flowering Arachis hypogaea L. and increase the a shoot number, thereby affecting other traits related thereto. 6. A construction method and use of a gene editing vector carrying a gene AhFH1 or an allele Ahfh1, wherein the gene editing vector is named KO-AhFH1; there are two target sequences for the construction method of the gene editing vector: sgRNA1 and sgRNA2 shown in SEQ ID NOs: 21-22; one of the two target sequences is ligated into an sgRNA region of a CRISPR/Cas9 vector BGKO41 to construct the gene editing vector KO-AhFH1 for the gene AhFH1; the gene editing vector is transformed into an alternate flowering Arachis hypogaea L. variety to change the gene AhFH1 through gene editing, and then defunctionalized offspring individuals are screened out to realize a change from alternate flowering Arachis hypogaea L. to continuous flowering Arachis hypogaea L., reducing a shoot number and increases a flower number, a pod number, and other related traits; and the sgRNA1 and the sgRNA2 are preferred target sequences, and a different target sequence is used according to a different CRISPR/Cas9 vector system or editing efficiency.
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