WO2022000835A1 - 花生开花习性基因AhFH1及其等位变异的克隆与应用 - Google Patents

花生开花习性基因AhFH1及其等位变异的克隆与应用 Download PDF

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WO2022000835A1
WO2022000835A1 PCT/CN2020/118264 CN2020118264W WO2022000835A1 WO 2022000835 A1 WO2022000835 A1 WO 2022000835A1 CN 2020118264 W CN2020118264 W CN 2020118264W WO 2022000835 A1 WO2022000835 A1 WO 2022000835A1
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ahfh1
gene
peanut
flowering
vector
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French (fr)
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张晓军
李季华
郭蕊
于晓娜
司彤
邹晓霞
王月福
王铭伦
迟晓元
禹山林
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青岛农业大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01H1/121—Plant growth habits
    • A01H1/1215—Flower development or morphology, e.g. flowering promoting factor [FPF]
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    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/54—Leguminosae or Fabaceae, e.g. soybean, alfalfa or peanut
    • A01H6/541—Arachis hypogaea [peanut]
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention belongs to the technical fields of plant molecular genetics and genetic engineering, and relates to the cloning and application of a peanut flowering habit gene AhFH1 (Flowering Habit 1 of Arachis hypogaea L.) and its allelic variation. Alternate-sequential flowering associated with flowering habit and the resulting traits of peanut branch number, pod number, pod concentration, ripening consistency, and pod yield are used for biotechnology genetic improvement or molecular breeding.
  • AhFH1 Food-s hypogaea L.
  • Alternate-sequential flowering associated with flowering habit and the resulting traits of peanut branch number, pod number, pod concentration, ripening consistency, and pod yield are used for biotechnology genetic improvement or molecular breeding.
  • Peanut (Arachis hypogaea L.) is rich in fat, protein and multivitamins, and has extremely high nutritional value, so it has become an important economic crop in many countries (Wan Shubo, 2003).
  • Peanut plant consists of an erect main stem, a pair of embryonic first side branches, multiple primary side branches developed at the base of the main stem, and secondary and tertiary secondary side branches developed on the side branches. .
  • the flowering habit of peanut is an important trait related to the plant type of peanut. Specifically, it is the differentiation problem from the development of peanut axillary bud primordium into inflorescence or branch, and the final manifestation is the arrangement of inflorescence and branch on the branch. There are two main types.
  • continuous flowering type ( Figure 1A) and alternating flowering type ( Figure 1B).
  • the most typical character of continuous flowering type is: the main stem blooms at the same time as each node (leaf axils) on the side branches can bear inflorescences, or on the base 1-2 nodes or the first node of the primary side branches, there are secondary vegetative branches. , inflorescences, all subsequent nodes have continuous inflorescences; on secondary branches, the first to second nodes and subsequent nodes are all inflorescences. This mode limits the number of branches of peanuts, so it is also called thin-branched peanuts.
  • the typical characters of the alternate flowering type are as follows: the main stem does not bloom, and the inflorescence and vegetative branches appear alternately on the side branches, generally vegetative branches grow on the 1-3 nodes or 1-2 nodes at the base of the side branches, no inflorescences, and 4 vegetative branches. -6- or 3-4-noded inflorescences, without vegetative branches, so alternate (common 2 flowers: 2 branches alternate).
  • This kind of peanuts start from the base with vegetative branches, and the branches occupy nearly half of the nodes. They have the characteristics of many branches and dense clusters, so they are also called dense branch peanuts.
  • the continuous flowering subspecies Aralpha beta subspecies
  • Alternaea subsp.hypogaea Alternas A, et al .2007.
  • there are less atypical or intermediate cultivars which mainly show continuous flowering with occasional branching and alternate branching while all leaf axils are flowering.
  • the flowering habit of peanut directly affects the above-ground plant type of peanut by affecting the number of inflorescences, the number of branches, the concentration of pods, and the consistency of maturity, which in turn affects the yield, planting method and kernel quality of peanuts. Because the development of peanut leaf axils proceeds gradually, the continuous flowering type of peanuts are more concentrated in time and space for flowering and pod formation, the maturity is more consistent, the pods are easy to harvest, and the quality is consistent. Alternate flowering peanuts had a large gap before and after flowering and pod formation, and the interval was far away, resulting in scattered pods, inconsistent pod ripening, and affected pod yield and quality.
  • Soybean is a photoperiod-sensitive short-day plant. It was found that GmFT1a, a member of the soybean FT gene family, can delay the flowering and maturation of soybean, and antagonizes the flowering-promoting genes GmFT2a/GmFT5a to jointly regulate the growth and development of soybean (Liu W, 2017, Kong F. , 2010, Sun H, 2011, Cai Y, 2018). Also as a typical short-day crop maize, the researchers cloned a quantitative trait locus (ZmCCT9) that controls the flowering period of maize through map-based cloning and association analysis. A Harbinger-like transposon upstream of this locus inhibits the expression of ZmCCT9 gene.
  • ZmCCT9 quantitative trait locus
  • the shoot apical meristem differentiates into the main stem of the plant, while the development of lateral branches is derived from the axillary meristem.
  • Environmental and plant internal factors are jointly regulated.
  • Many genes related to the control of branch development have been obtained in the study of branching patterns of plants such as Arabidopsis, rice, tomato, and maize (Zhi WNT, 2014, Soyk S., 2017). These genes can be divided into two categories according to the branching phenotype of plants. One is the gene that controls the formation of leaf axillary meristems, such as the LS gene first discovered in tomato.
  • This gene can not only control the formation of axillary meristems, but also ls
  • the mutant tomato plants showed almost no branch formation (Schumacher K, 1999), and the LS homologous gene OsMOC1 gene was also found in rice. , 2003), in addition to regulate the BL gene of axillary meristem in tomato (Gregor S, 2002), and found the homologous gene RAX of tomato BL in Arabidopsis (Keller T, 2006);
  • Axillary meristem growth-related genes in which these mutations do not affect axillary meristem formation include the TB1 gene in maize that inhibits axillary bud growth (Doebley J, 1997, Lauren H, 2002), and the TB1 gene in rice.
  • the source gene is called OsTB1 or FINECULM1 and is called BRC1 in Arabidopsis, pea and tomato (Aguilar-Mart ⁇ nez JA, 2007, Nils B, 2012, Mar MT, 2011, Minakuchi K, 2010), in Arabidopsis thaliana
  • the BRC1 gene which encodes a protein similar to TB1, regulates axillary bud development in Arabidopsis (Aguilar-Mart ⁇ nez JA, 2007), and SlBRC1a and SlBRC1b genes with similar functions to the Arabidopsis BRC1 gene were found in tomato (Mar MT, 2011) and so on. Recently, it has also been reported that strigolactone regulates Arabidopsis collateral development through BRC1 (Wang, L., et al. 2020).
  • the peanut flowering habit gene At present, there are few researches on the peanut flowering habit gene. Although there are bioinformatics analysis of the peanut florigen gene family, at least 29 members of the florigen homologous gene family of cultivated peanut are predicted. Specifically, It is not clear which gene controls alternating consecutive flowering in peanut (Jin, Tang et al. 2019). The cloning and functional study of peanut flowering habit gene has not been reported yet. Locating and cloning the peanut flowering habit gene will provide target genes for genetic improvement and genetic engineering or gene editing breeding for peanut flowering habit and related traits.
  • the present invention provides the cloning and application of a peanut flowering habit gene AhFH1 and its allelic variation.
  • the present invention provides the cloning and application of a peanut flowering habit gene AhFH1 and its allelic variation.
  • the genetic segregation population constructed by the hybridization of alternate flowering peanut varieties and continuous flowering peanut varieties is combined with candidate candidates between parents through linkage positioning and map-based cloning.
  • the gene sequence difference analysis identified the candidate gene AhFH1 (as shown in Figure 2).
  • the cloning, comparative analysis and association verification of the gene AhFH1 in germplasm resources found that there are at least three allelic variations in the peanut flowering habit gene AhFH1: one complete A functional allelic variant, AhFH1, and two defunctionalized allelic variants, Ahfh1 (including both defunctionalized allelic variants, Ahfh1-1 and Ahfh1-2).
  • the present invention provides the application of the above-mentioned gene AhFH1 and its allelic variation and promoter in crop genetic improvement, preferably in the flowering habit of peanut and the number of branches, pod number, pod concentration, maturity consistency and pod caused by it. Applications in molecular genetic improvement of traits such as yield.
  • the nucleotide sequence of the peanut flowering habit gene AhFH1 of the present invention is SEQ ID NO: 1 at the genome level
  • the cDNA sequence corresponding to the transcribed mRNA is SEQ ID NO: 2
  • the encoded protein sequence is SEQ ID NO: 2 :3.
  • the representative varieties of allelic variation AhFH1 are the peanut genome sequencing variety Tifrunner or Zhejiang local variety Xiaohongmao, etc.
  • the corresponding peanut flowering habit is alternate flowering type.
  • the cloned primer pair of the described peanut flowering habit gene AhFH1 at the genome level is FH1g-F/R, and its nucleotide sequence is such as SEQ ID NO: 4-5, and the electrophoresis diagram of the cloned product in the representative variety using this primer pair is shown in the figure. 3; the clone primer pair FH1cd-F/R of peanut flowering habit gene AhFH1 at its cDNA level, its nucleotide sequence is such as SEQ ID NO: 6-7, use this primer pair to clone complete functionality in the cDNA of the representative variety, etc.
  • the complete coding frame of the position variant AhFH1, and its product electropherogram is shown in Figure 4.
  • the defunctionalized allelic variation Ahfh1-1 of the present invention has a nucleotide sequence at the genome level such as SEQ ID NO: 8, and the defunctionalized allelic variation Ahfh1-1 is that there is an ATG at the end of the gene at the genome level. It is the deletion of 1492bp from the position of +1872bp to +3273bp (the deletion is named as the functional molecular marker InDel-1492bp), which contains most and all of the 3'UTR of the last exon.
  • the representative cultivars are the genome sequencing cultivar Shitouqi and the local cultivar Fu peanut, etc.
  • the flowering habit of the corresponding peanut is continuous flowering type.
  • the defunctionalized allelic variation Ahfh1-2 of the present invention has a nucleotide sequence at the genome level such as SEQ ID NO: 11, and the base C at the +335bp position of the cDNA encoded by the defunctionalized allelic variation Ahfh1-2 Deletion, the encoded cDNA sequence is such as SEQ ID NO: 12, the deletion of the base C causes the frameshift of the translation frame of Ahfh1-2, and the terminator is formed in advance, and the translated protein is incomplete and non-functional.
  • the representative varieties of this allelic variation are the peanut varieties Yunnan Qicai and Longhuan 559, etc.
  • the flowering habit of the corresponding peanut is continuous flowering type.
  • the clone primer pair FH1g-F/R (SEQ ID NO:4-5) of the above-mentioned gene AhFH1 genome level and the clone primer pair FH1cd-F/R (SEQ ID NO:6-7) of the cDNA level can also be achieved respectively.
  • the invention also provides a functional molecular marker InDel-1492bp for distinguishing the allelic variation of the alternate flowering type AhFH1 and the continuous flowering type Ahfh1-1 of the peanut flowering habit gene, and the corresponding primer pair is InDel-1492bp-F/R, which The nucleotide sequence is shown in SEQ ID NO: 9-10 (this primer pair is the preferred primer, other primer pairs that can amplify and identify the 1492bp deletion between the above-mentioned AhFH1 and Ahfh1-1 can also be selected).
  • the amplification product of the functional molecular marker InDel-1492bp can be detected by agarose electrophoresis to distinguish the two allelic variations of AhFH1 and Ahfh1-1.
  • the amplification product of AhFH1 is 2556bp
  • the amplification product of Ahfh1-1 The amplified product is 1064bp (as shown in Figure 5).
  • the invention also provides the application of a promoter sequence regulating the peanut flowering habit gene AhFH1 in crop genetic improvement, preferably in improving the flowering habit of peanut and its related branch number, pod number, pod concentration, maturity consistency and Application in traits such as pod yield;
  • the promoter sequences of the gene AhFH1/Ahfh1 have two main types, respectively from Tifrunner and Lionhead Enterprise, respectively as shown in SEQ ID NOs: 13-14, while providing promoter clones
  • the primer pair is FH1p-F/R, and its nucleotide sequence is SEQ ID NO:15-16. Using this primer pair, the promoter of the gene AhFH1 can be cloned.
  • the latter mainly has a 214bp insertion (named as molecular marker InDel-214bp), and this difference can be detected by agarose electrophoresis (Fig. 6).
  • the representative species can be expanded into three types of belts, in addition to the Tifrunner type with a single short belt type and the lion head type with a single long belt type, and the double belt type Florunner with both long and short belts, the double belt type is four times the heterologous type.
  • the two subgenomic homologous genes of the A and B subgenes of somatic peanut are without and with 214bp insertion, respectively.
  • the molecular marker InDel-214bp can be used for molecular marker-assisted selection of the AhFH1 gene locus of the progeny of biparental hybridization.
  • the present invention also provides an overexpression recombinant construct: an overexpression vector p35S::AhFH1 using the 35S promoter of tobacco mosaic virus, the vector backbone of which is a plant overexpression vector PHB, which contains the peanut flowering habit gene AhFH1 Relevant nucleotide sequences, the primer pairs required for the construction of the overexpression vector are OE-FH1-F and OE-FH1-R, and their sequences are shown in SEQ ID NOs: 17-18; this primer pair is used in alternate flowering peanuts
  • the gene AhFH1 is obtained by amplification in the cDNA of the cDNA or in the plasmid containing the complete coding frame of the gene, and the amplified product is connected by enzyme cutting or recombination into the overexpression vector PHB (as shown in Figure 7) or other plant overexpression vectors, and is constructed into a The species overexpressed the transgenic vector p35S::AhFH1 ( Figure 7A). Transforming the over
  • the present invention also provides a complementary expression recombination construct: based on the above-constructed overexpression transgenic vector p35S::AhFH1, using the self-promoter of the gene AhFH1 to construct a complementary expression transgenic vector: pFH1::AhFH1, which contains the peanut Nucleotide sequences related to flowering habit gene AhFH1.
  • the primer pair required for the construction of the complementary expression vector is FH1pro-F/R, the sequence of which is shown in SEQ ID NO: 19-20, the EcoR I restriction site of the upstream primer FH1pro-F is " gaattc ", and the downstream primer The Pst I restriction site of FH1pro-R is " ctgcag ".
  • the primers were used to clone and amplify the DNA of the alternate flowering peanut varieties, and the amplified product or the amplified product of the T vector was directly connected to the target fragment recovered by EcoR I and Pst I double enzyme digestion into the same enzyme.
  • the large fragment recovered from the linearized overexpression transgenic vector p35S::AhFH1 was constructed into a complementary expression transgenic vector pFH1::AhFH1 (as shown in Figure 7B).
  • the complementary expression vector can also be constructed by using appropriate primers to directly amplify the full-length genome of the functional AhFH1 including the promoter and coding region in the alternate flowering cultivar into an appropriate plant transgenic vector, which will not be repeated here. Transforming the complementary expression vector into continuous flowering peanut can change it into alternate flowering peanut, increase the number of branches, and then affect other related traits.
  • the present invention also provides a gene editing vector construct, which contains the AhFH1 or a partial nucleotide sequence of the allele Ahfh1 described in the present invention, and the vector is the gene editing vector KO-AhFH1.
  • the vector is the gene editing vector KO-AhFH1.
  • the sgRNA regions of BGK041 (Fig. 8) were respectively constructed into gene editing knockout vector KO-AhFH1 targeting the target gene AhFH1.
  • Transforming the gene editing vector into alternate flowering peanut varieties can change the AhFH1 gene by gene editing, and then select functionally edited progeny, and realize the conversion of alternate flowering peanuts to continuous flowering, reducing the number of branches and increasing the number of branches.
  • the sgRNA1 and sgRNA2 are preferred target sequences, and the target sequences can be different according to different CRISPR/Cas9 vector systems or editing efficiencies.
  • the peanut flowering habit gene AhFH1 and its allelic variation of the present invention are directly derived from peanuts, and can also be derived from homologous genes with sufficiently high similarity in crops such as soybean, rape, cotton, rice, corn, and wheat.
  • the present invention also provides a method for improving the flowering habit of peanut, the method comprising preparing a peanut plant containing the above-mentioned AhFH1 or allele Ahfh1-related nucleotide sequence construct.
  • the cloning and application of the peanut flowering habit gene AhFH1 provided by the present invention has the following beneficial effects:
  • the peanut flowering habit gene AhFH1 provided by the invention and its allelic variation are for exploring the molecular mechanism of the peanut flowering habit gene AhFH1 regulating peanut flowering habit and preliminarily constructing its molecular network participating in the regulation of flowering and branching, and studying the function of this gene It provides important reference for the evolution law among crops.
  • the difference between the peanut flowering habit gene AhFH1 and its allelic variant genes provided by the present invention can be developed as a functional molecular marker for crop molecular marker-assisted selection breeding, preferably in improving the flowering habit of peanut and its related
  • the number of branches, the number of pods, the concentration of pods, the consistency of ripening and the yield of pods play a key role.
  • the gene sequence and amino acid or polypeptide or protein of the peanut flowering habit gene AhFH1 provided by the present invention are in crop genetic improvement, preferably in improving the flowering habit of peanut and the number of branches, pod number, pod concentration, maturity It plays a key role in traits such as consistency and pod yield.
  • Fig. 1 is a schematic diagram of the flowering habit of peanuts involved in the present invention, A is a continuous flowering type, and B is an alternate flowering type.
  • Figure 2 is a diagram of the map-based cloning process of the peanut flowering habit gene AhFH1 involved in the present invention.
  • Figure 3 is an electrophoresis diagram of the full-length clone of the peanut flowering habit gene AhFH1 involved in the present invention at the genome level of representative peanut varieties (primer pair FH1g-F/R).
  • Figure 4 is an electrophoresis diagram of the cDNA clone of the peanut flowering habit gene AhFH1 involved in the present invention (primer pair FH1cd-F/R).
  • Figure 5 is an electrophoresis diagram of InDel-1492bp, a functional molecular marker for identifying two allelic variations of AhFH1 and Ahfh1-1 involved in the present invention (primer pair InDel-1492bp-F/R).
  • Figure 6 is an electrophoresis diagram of the two promoters of the peanut flowering habit gene AhFH1 involved in the present invention cloned in the genome (primer pair FH1p-F/R).
  • Figure 7 is a structural diagram of the constructs p35S::AhFH1 and pFH1::AhFH1 involved in the present invention.
  • Figure 8 is a structural diagram of the gene editing construct KO-AhFH1 involved in the present invention.
  • Example 1 Map-based cloning of the peanut flowering habit gene AhFH1
  • Transcriptome sequencing was performed on Pingdu 9616 and Florunner and 60 individuals (30 alternate flowering types and 30 continuous flowering types), respectively, and the transcriptome sequencing data of 62 samples were obtained.
  • the SNP results were screened by comparing the transcriptome data table with the reference genome sequence of the cultivar Tifrunner, and finally 12,421 high-quality credible SNP loci were obtained.
  • the high-quality SNPs were analyzed by ⁇ SNP-index between alternate flowering ponds and continuous flowering ponds, and the flowering habit gene was initially located at the end of chromosome 12, that is, between 117 682 534 bp and 119 846 824 bp of chromosome 12.
  • the length is approximately 2.16M (Tifrunner reference genome, first edition).
  • Arahy.BBG51B was preliminarily selected as the candidate gene of the peanut flowering habit gene AhFH1 by fine mapping.
  • the sequence alignment of this candidate gene between the reference genomes Tifrunner (alternate flowering type) and lion head (continuous flowering type) found that there is a 214bp in the promoter region of the candidate gene of lion head enterprise (continuous flowering type). Insertion, while the reference sequence of the coding region of lion's head is incomplete.
  • the clone primer FH1g-F/R (SEQ ID NO: 4-5) for the gene AhFH1 genome level was designed, using this primer pair to take the genomic DNA of alternate flowering peanut as a template, and clone the described by PCR amplification
  • the complete genome sequence of the candidate gene ( Figure 3).
  • the primer pair FH1cd-F/R for cloning the gene AhFH1 from cDNA was designed, and its sequence is shown in SEQ ID NO: 6-7, and the primer pair was used to alternate the cDNA of the stem end of the side branch or the leaf tissue of flowering peanut.
  • primer pair FH1g-F/R In the amplification of the full-length gene (primer pair FH1g-F/R), there are indeed representative cultivars that have not cloned the full-length gene at the genome level, so continue to design primer pairs with a larger span downstream of the candidate gene,
  • the primer pair can amplify the allelic variation Ahfh1-1 (SEQ ID NO: 8) corresponding to the smaller fragment.
  • the amplified fragment was sequenced and found to have a 1492bp deletion.
  • This primer pair was named InDel- 1492bp-F/R, whose sequence is shown in SEQ ID NO: 9-10, can directly identify two allelic variants of AhFH1 and Ahfh1-1, and the amplification product corresponding to the allelic variant AhFH1 is 2556bp, corresponding to the allelic variant Ahfh1-
  • the amplification product of 1 is 1064bp, and the difference can be detected by agarose electrophoresis (as shown in Figure 5).
  • This molecular marker can be used to carry out molecular marker-assisted selection of allelic variation of flowering habit in cross-breeding between varieties with allelic differences of AhFH1 and Ahfh1-1, or to perform allelic variation of AhFH1 and Ahfh1-1 in germplasm resources. identify.
  • the cloning primer FH1p-F/R (SEQ ID NO: 15-16) for the promoter was designed according to the reference sequence, the candidate gene AhFH1 promoter was cloned in representative varieties, and the target band was sequenced and analyzed.
  • the comparative analysis of cloning and sequencing it was found that there are two main types of promoter sequences of the candidate gene AhFH1, respectively from Tifrunner and Lionhead Enterprise (SEQ ID NO: 13-14), but there are three types of cloned strips in the germplasm, In addition to the single Tifrunner-type short band and the lion-head enterprise-type long band, there are also double-banded types with two long and short bands (as shown in Figure 6). Combined with the analysis of later research results, it is found that the double-banded type is allotetraploid peanut A and Both promoter types are present in both sets of B subgenes.
  • the insertion of the promoter region (214 bp) and the deletion of the gene end (1492 bp) found by sequencing and comparison of representative cultivated peanut varieties may affect the continuous flowering type and alternating flowering type of peanut.
  • the insertion of the promoter region (214 bp) and the deletion of the gene end (1492 bp) were developed as InDel markers, defined as FH1p-F/R and InDel-1492 bp, respectively. Correlation verification was carried out in 268 cultivar germplasm resources with rich flowering habit. It was found that except for the insertion of the promoter region (214bp), the correlation between the phenotype and the 1492bp deletion type at the end of the gene was all continuous flowering type.
  • the candidate gene Arahy.BBG51B as the peanut flowering habit gene AhFH1 (Flowering Habit 1 of Arachis hypogaea L.), and the gene AhFH1 has a complete functional allelic variation AhFH1 and at least two defunctionalizations, etc. Positional variants Ahfh1-1 and Ahfh1-2.
  • the homologous chromosomes A02 and B02 of different sets of chromosomes are almost identical in the range of about 500kb upstream and downstream of this candidate gene, which may be the A and B subgenomes Arahy.DYRS20 annotated on the genome of A02 is exactly the same as Arahy.BBG51B, so the AhFH1 described in this example contains two loci: Arahy.DYRS20 of the A02 chromosome (named AhFH1A) Arahy.BBG51B with chromosome B02 (designated it AhFH1B).
  • the peanut flowering habit gene AhFH1 theoretically has four genotypes in the allotetraploid cultivated peanut composed of two sets of subgenomes A and B: AhFH1A/AhFH1B, Ahfh1a/Ahfh1b, Ahfh1a /AhFH1B and AhFH1A/Ahfh1b, and usually in the same variety, A is exactly equal to B, and a is exactly equal to b, so it can be simply divided into three genotypes: AhFH1/AhFH1, Ahfh1/Ahfh1 and AhFH1/Ahfh1 , among which AhFH1/AhFH1 and AhFH1/Ahfh1 are alternate flowering genotypes, and only Ahfh1/Ahfh1 are continuous flowering genotypes.
  • the 35S of tobacco mosaic virus was used as the promoter to construct the overexpression transgenic vector p35S::AhFH1, and the mRNA of the peanut flowering habit gene AhFH1 was overexpressed in the continuous flowering cultivar (Flower 23) by pollen tube introduction.
  • the specific steps include: using Sac I and Xba I double digestion to cut the GFP on the overexpression vector PHG, using the T plasmid as a template to utilize the primers OE-AhFH1-F and OE-AhFH1-R of homologous recombination, and its sequence As shown in SEQ ID NOs: 17-18, the target fragment is amplified, the target fragment amplified by the T plasmid and the PHB backbone fragment of the overexpression vector are recovered by gel, and the ligation is carried out by homologous recombination after purification, and the ligation product is heat-shocked.
  • Coliform DH5a competent cells were coated with kanamycin-resistant LB plates, and single clones were picked and tested by PCR. The positive clones were sent to Qingdao Qingke Zixi Biotechnology Co., Ltd. for sequencing, and the strains with correct sequencing were selected.
  • bacteria extract the plasmid with the target fragment, which is the AhFH1 overexpression transgenic vector: p35S::AhFH1, the structure of which is shown in Figure 7A.
  • the AhFH1 overexpression vector was transformed into Agrobacterium GV3101 competent cells, coated with kanamycin and rifampicin double antibody YEB plates, and single clones were picked and tested positive by PCR as transgenic strains for later use. Transforming the overexpression transgene vector into continuous flowering peanut can increase the number of branches, which in turn affects other traits associated with it.
  • the primer pairs required for the construction of the complementary expression transgenic vector are FH1pro-F and FH1pro-R, the sequences of which are shown in SEQ ID NOs: 19-20, and the primer pairs are used to clone and amplify in the DNA template of alternate flowering peanuts,
  • the amplification product of the connected T vector or the target fragment recovered by double digestion with EcoR I and Pst I was directly connected into the overexpression transgenic vector p35S::AhFH1 linearized by the same digestion, and constructed into a Complementary expression of the transgenic vector pFH1::AhFH1 ( Figure 7B).
  • FH1pro-F 5'-CG GAATTC ACGAAATCTCAACTTGTTTACGT-3' (SEQ ID NO: 18)
  • FH1pro-R 5 ⁇ -AA CTGCAG TGTTAAAGAGAATGAAAGAGAA-3 ⁇ (SEQ ID NO: 19); (FH1pro primer: EcoR I restriction site upstream of AhFH1pro-F is "GAATTC”, FH1pro-R downstream of the Pst I digested The site is "CTGCAG").
  • the construction of the complementary expression transgenic vector can also use appropriate primers to directly amplify the full-length genome of the functional AhFH1 including the promoter and coding region in the alternate flowering cultivar and connect it to an appropriate plant transgenic vector, which will not be repeated here.
  • the self-promoter of the peanut flowering habit gene AhFH1 is used as the promoter to construct an overexpression vector, and the mRNA of the peanut flowering habit gene AhFH1 is overexpressed in the continuous flowering type variety (Flower 23) by the pollen tube introduction method, and the specific steps include:
  • the self-promoter expression vector was constructed based on the constructed 35S promoter overexpression vector p35S::AhFH1; the overexpression vector p35S::AhFH1 was double digested with EcoR I and Pst I to excise the 35S promoter sequence, and the overexpression vector was recovered.
  • the large fragment of the vector p35S::AhFH1 is about 12kbp; the primer pair is FH1pro-F/R, the sequence of which is shown in SEQ ID NO: 19-20, and the promoter of the AhFH1 gene of the cultivated peanut red hair was cloned and combined with T
  • the vector was ligated and transformed, sequenced to extract the plasmid, and then the extracted plasmid was double digested with EcoR I and Pst I to recover the target fragment, and then the recovered overexpression vector p35S::AhFH1 large fragment and T4 ligase were used.
  • the target fragment amplified by EcoR I and Pst I double-enzyme digestion and recovered from the vector was connected and transformed into E.
  • the complementary expression vector pFH1::AhFH1 of the AhFH1 self-promoter and its structure is shown in Figure 7B.
  • the overexpression vector of AhFH1 self-promoter was transformed into Agrobacterium GV3101 competent cells, coated with YEB plates with dual antibodies to kanamycin and rifampicin, and single clones were picked and tested positive by PCR as transgenic strains for future use. Transforming the complementary expression vector into continuous flowering peanut can change it into alternate flowering peanut, increase the number of branches, and then affect other related traits.
  • the CRISPR/Cas9 system is used to perform gene editing and knockout.
  • the specific operation steps include: online design and generation of sgRNA target sequences (http://www.biogle.cn/index/excrispr), and selection of two targets with the highest scores Site sgRNA1 and sgRNA2 (SEQ ID NO: 21-22), the generated sgRNA sequence was handed over to Qingdao Qingke Zixi Biotechnology Co., Ltd.
  • the Oligo dimer is connected to the linearized CRISPR/Cas9 vector by ligase to be the KO-AhFH1 vector; 2ul KO-AhFH1 vector, 1ul Oligo dimer, 1 ⁇ l Enzyme Mix and 16ul ddH were added to a 200ul PCR tube 2 O, after mixing, react at room temperature (20 °C) for 1 hour, transform the ligation products into E. coli DH5a competent cells by heat shock method, spread kanamycin-resistant LB plates, pick out single clones, and use PCR After testing, the positive clones were sent to Qingdao Qingke Zixi Biotechnology Co., Ltd.
  • the strains with correct sequencing were selected, and the plasmid was extracted, which is the AhFH1 knockout plasmid: KO-AhFH1-1/2.
  • the AhFH1 gene knockout plasmid KO-AhFH1-1/2 was transformed into Agrobacterium competent cells, coated with kanamycin and rifampicin double-antibody YEB plates, and single clones were picked and tested positive by PCR, and positive clones were selected for transformation Alternate flowering peanuts (such as small red hair or 209 small peanuts); the CRISPR/Cas9 vector selects BGK041 (as shown in Figure 8), which uses the soybean U6 promoter to drive the sgRNA sequence, which can be efficiently used in dicotyledonous plants.
  • the enhanced CaMV 35S promoter expresses the Cas9 protein efficiently.
  • Transforming the gene editing vector into alternate flowering peanut varieties can change the AhFH1 gene by gene editing, and then select functionally edited progeny, and realize the conversion of alternate flowering peanuts to continuous flowering, reducing the number of branches and increasing the number of branches. The number of flowers, the number of pods and other traits related to it.
  • the CRISPR/Cas9 vector BGK041 backbone used for gene editing was purchased from Biogle Gene (http://www.biogle.cn/index/excrispr), which is only for illustration, and other plant CRISPR/Cas9 gene editing vectors or Other single base editing vectors.

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Abstract

提供了一种花生开花习性基因AhFH1及其等位变异基因在改良花生的开花习性相关性状中的应用，该AhFH1基因的核苷酸序列如SEQ ID NO:1所示，去功能性等位变异基因Ahfh1-1的核苷酸序列如SEQ ID NO:8所示，去功能性等位变异基因Ahfh1-2的核苷酸序列如SEQ ID NO:11所示。还提供了AhFH1基因的克隆引物，区分花生开花习性基因完整功能型AhFH1和去功能性Ahfh1-1的功能性分子标记InDel-1492bp，含有AhFH1基因的过表达转基因载体、互补表达转基因载体和基因编辑载体的构建方法及应用。

Description

花生开花习性基因AhFH1及其等位变异的克隆与应用 技术领域：
本发明属于植物分子遗传学和基因工程技术领域，涉及花生开花习性基因AhFH1(Flowering Habit 1 of Arachis hypogaea L.)及其等位变异的克隆与应用，通过分子生物学手段利用该基因可以对花生开花习性相关的交替-连续开花及由其引起的花生分枝数目、荚果数目、荚果集中程度、成熟一致性和荚果产量等性状进行生物技术的遗传改良或分子育种。
背景技术：
花生(Arachis hypogaea L.)富含脂肪、蛋白质和多种维生素，营养价值极高，因此其成为了许多国家重要的经济作物(万书波，2003)。花生植株由一条直立的主茎、一对胚起源的第一侧枝和发育于主茎基部的多条一级侧枝和侧枝上又发育的二级、三级等次级侧枝构成的束状株型。花生的开花习性是花生株型相关的一个重要的性状，具体是由花生腋芽原基发育成花序或者枝条的分化问题，最终表现为花序和分枝在枝条上的排列方式问题，主要有两种典型的代表：连续开花型(如图1A)和交替开花型(如图1B)。连续开花型最典型的性状表现为：主茎开花同时侧枝上每一节(叶腋)均可着生花序，或在一级侧枝的基部1-2节或第一节上着生二次营养枝、花序，以后各节全部连续着生花序；在二级分枝上第一至第二节及以后各节均着生花序。这种模式限制了花生的分枝数，所以又称疏枝型花生。交替开花型的典型性状表现为：主茎不开花同时花序与营养枝在侧枝上交替出现，一般是侧枝基部1-3节或1-2节上生长营养枝，不长花序，其后的4-6节或3-4节长花序，不长营养枝，如此交替发生(常见2花:2枝交替出现)。这类花生从基部开始着生营养枝，且分枝占节位的近二分之一，有分枝多而密的株丛特点，所以又称密枝型花生。分类学上简单地根据主茎是否开花将花生栽培品种分为两个亚种：连续开花亚种(Arachis hypogaea subsp.fastigiata)和交替开花亚种(Arachis hypogaea subsp.hypogaea)(Krapovickas A,et al.2007)。除此之外还有较少的非典型性或中间型品种，主要表现为连续开花偶有分枝和交替分枝同时所有叶腋均开花等类型。花生的开花习性通过影响花生的花序数目、分枝数目和荚果集中程度、成熟一致性等直接影响花生地上部株型，进而影响花生群体产量、种植方式和籽仁的品质。由于花生叶腋发育渐次进行，因而连续开花型花 生开花结荚时间和空间上均比较集中、成熟较一致、荚果收获容易、品质一致。交替开花型花生开花结荚时间上前后差距较大，间隔较远，导致荚果分散、荚果成熟不一致、荚果的产量和品质均受到影响。
目前在拟南芥、番茄、大豆、水稻和玉米等植物中，针对花序分化与发育的遗传基础研究已经取得了较大的进展。TFL1同源基因和FT同源基因LFY/AP1之间的相互拮抗共同调节拟南芥花序分枝模式(Alvarez J，1992；Liljegren SJ，1999；Conti L，2007；Hiraoka K，2013)。拟南芥tfl1突变体具有单花，并导致花序分枝中花序顶端分生组织终止为单独的花(Alvarez J，1992)；TFL1过表达导致拟南芥晚开花(Benlloch R，2007)。大豆作为光周期敏感的短日照植物，研究发现大豆FT基因家族成员GmFT1a能够延迟大豆开花和成熟，与开花促进基因GmFT2a/GmFT5a相互拮抗，共同调节大豆的生长发育(Liu W，2017，Kong F.，2010，Sun H，2011，Cai Y，2018)。同样作为典型的短日照作物玉米，研究者通过图位克隆和关联分析克隆到一个控制玉米开花期的数量性状位点(ZmCCT9)，该位点上游的一个Harbinger-like转座子抑制ZmCCT9基因的表达，从而促进玉米在长日照条件下开花(Huang C，2017，Hsiao-Yi H，2012，Yang Q，2013)。在番茄中研究发现局部FT(特别是SFT)和TFL1(SP)的比例控制初级枝条和次生枝条有限或无限生长的平衡。番茄的枝桠结构随成花素(sft)和成花素抑制基因(sp)增减而发生的变化，因此利用杂交和基因编辑手段培育出高产的番茄品种，为获得理想型植株提供了新的研究方向(Krieger U.，2010，Soyk S.，2017)。
在植物的分枝发育过程中，茎尖分生组织(SAM)分化成植物的主茎，而侧枝的发育则是由腋生分生组织分化而来，其中腋生分生组织分化为侧枝的过程受环境和植物内部因素共同调控。在拟南芥、水稻、番茄和玉米等植物的分枝模式研究中获得了很多与控制分枝发育相关的基因(Zhi WNT，2014，Soyk S.，2017)。根据植物的分枝表型可将这些基因分为两类，一类为控制叶腋分生组织形成的基因，如最早在番茄上发现的LS基因，该基因不仅可以控制腋生分生组织形成，ls突变体的番茄植株表现为几乎不形成分枝(Schumacher K，1999)，在水稻中也发现了LS的同源基因OsMOC1基因，突变后导致水稻不能形成分蘖芽从而影响水稻的分蘖数目(Li X，2003)，另外在番茄中调控腋生分生组织的BL基因(Gregor S，2002)，并在拟南芥中发现了番茄BL的同源基因RAX(Keller T，2006)；另一 类是与腋生分生组织生长相关的基因，其中这些突变并不影响腋生分生组织的形成，其中包括玉米中与抑制腋芽生长的TB1基因(Doebley J，1997，Lauren H，2002)，水稻中的TB1同源基因被称为OsTB1或FINECULM1，拟南芥、豌豆和番茄中被称为BRC1(Aguilar-Martínez JA，2007，Nils B，2012，Mar MT，2011，Minakuchi K，2010)，在拟南芥中与TB1编码类似蛋白的BRC1基因，其调控拟南芥的腋芽发育(Aguilar-Martínez JA，2007)，并在番茄中发现与拟南芥BRC1基因具有类似的功能的SlBRC1a和SlBRC1b基因(Mar MT，2011)等。最近也有报道独角金内酯通过BRC1调控拟南芥的侧枝发育(Wang,L.,et al.2020)。
目前对于花生开花习性基因的研究工作较少，虽然有对花生成花素基因家族开展的生物信息学分析，但栽培花生的成花素同源基因家族成员预测的至少有29个以上，具体是哪一个基因控制了花生的交替连续开花并不清楚(Jin,Tang et al.2019)。对花生开花习性基因的克隆和功能研究还未见报道，定位并克隆花生开花习性基因，将为针对花生的开花习性及其相关性状进行的遗传改良和基因工程或基因编辑育种提供目标基因。
发明内容：
为了克服上述缺陷，本发明提供一种花生开花习性基因AhFH1及其等位变异的克隆与应用。
为了达到上述目的，本发明的技术方案是：
本发明提供一种花生开花习性基因AhFH1及其等位变异的克隆与应用，利用交替开花花生品种与连续开花花生品种杂交构建的遗传分离群体，通过连锁定位和图位克隆，结合亲本间的候选基因序列差异分析，明确了候选基因AhFH1(如图2)，对基因AhFH1在种质资源中的克隆、比较分析和关联验证等发现花生开花习性基因AhFH1至少存在三种等位变异：一种完整功能性等位变异AhFH1和两种去功能性的等位变异Ahfh1(同时包括去功能性等位变异Ahfh1-1和Ahfh1-2)。本发明提供对上述基因AhFH1及其等位变异和启动子在作物遗传改良中的应用，优选在花生的开花习性及由其引起的分枝数目、荚果数目、荚果集中程度、成熟一致性和荚果产量等性状的分子遗传改良中的应用。
本发明所述的花生开花习性基因AhFH1，其基因组水平的核苷酸序列为SEQ ID NO:1，其转录的mRNA对应的cDNA序列为SEQ ID NO:2，其编码的蛋白序列为SEQ ID NO:3。等位变异AhFH1的代表品种为花生基因组测序品种 Tifrunner或浙江地方品种小红毛等，其对应花生的开花习性为交替开花型。所述花生开花习性基因AhFH1在基因组水平的克隆引物对为FH1g-F/R，其核苷酸序列如SEQ ID NO:4-5，利用该引物对在代表品种中的克隆产物电泳图如图3；花生开花习性基因AhFH1在其cDNA水平的克隆引物对FH1cd-F/R，其核苷酸序列如SEQ ID NO:6-7，利用该引物对在代表品种的cDNA中克隆完整功能性等位变异AhFH1的完整编码框，其产物电泳图如图4。
本发明所述的去功能性等位变异Ahfh1-1，其基因组水平的核苷酸序列如SEQ ID NO:8，去功能性等位变异Ahfh1-1是在基因组水平的基因末端存在一个以ATG为起始的+1872bp至+3273bp位置的1492bp的缺失(将该缺失命名为功能性分子标记InDel-1492bp)，它包含最后一个外显子大部分及全部的3`UTR，该等位变异的代表品种为基因组测序品种狮头企和地方品种伏花生等，其对应花生的开花习性为连续开花型。
本发明所述的去功能性等位变异Ahfh1-2，其基因组水平的核苷酸序列如SEQ ID NO:11，去功能性等位变异Ahfh1-2编码的cDNA的+335bp位置的碱基C缺失，其编码的cDNA序列如SEQ ID NO:12，该碱基C缺失导致Ahfh1-2的翻译框移码，并提前形成终止子，翻译的蛋白不完整无功能。该等位变异的代表品种为花生品种云南七彩和龙花生559等，其对应花生的开花习性为连续开花型。上述基因AhFH1基因组水平的克隆引物对FH1g-F/R(SEQ ID NO:4-5)和cDNA水平的克隆引物对FH1cd-F/R(SEQ ID NO:6-7)，同样可以分别实现对Ahfh1-2基因组水平和cDNA水平的克隆。基因AhFH1和等位变异Ahfh1-2间的单碱基差异可以通过对扩增产物的测序进行区分。
本发明还提供一种区分花生开花习性基因的交替开花型AhFH1和连续开花型Ahfh1-1等位变异的功能性分子标记InDel-1492bp，其对应的引物对为InDel-1492bp-F/R，其核苷酸序列如SEQ ID NO：9-10所示(该引物对为优选引物，其他可以扩增鉴别上述AhFH1和Ahfh1-1间1492bp缺失的引物对亦可选用)。结合测序确认AhFH1序列无变异的情况下，功能性分子标记InDel-1492bp扩增产物通过琼脂糖电泳检测可以区分AhFH1和Ahfh1-1两种等位变异，AhFH1的扩增产物为2556bp，Ahfh1-1的扩增产物为1064bp(如图5)。
本发明还提供一种调控花生开花习性基因AhFH1的启动子序列在作物遗传 改良中的应用，优选在改良花生的开花习性及与其相关的分枝数目、荚果数目、荚果集中程度、成熟一致性和荚果产量等性状中的应用；所述基因AhFH1/Ahfh1的启动子序列有两种主要分型，分别来自Tifrunner和狮头企，分别如SEQ ID NO:13-14所示，同时提供启动子克隆引物对为FH1p-F/R，其核苷酸序列为SEQ ID NO:15-16。利用该引物对可以对基因AhFH1的启动子进行克隆。比较Tifrunner和狮头企，后者主要存在一个214bp的插入(将其命名为分子标记InDel-214bp)，通过琼脂糖电泳可以检测这一差异(如图6)。代表品种可以扩增出三种带型，除了单条短带型的Tifrunner型和单条长带型的狮头企型，还有长短带都有的双带型Florunner，双带型为异源四倍体花生的A和B亚基因的两个亚基因组同源基因分别为无214bp插入和有214bp插入。分子标记InDel-214bp可以用于对双亲杂交后代的AhFH1基因位点的分子标记辅助选择。
本发明还提供了一种过表达重组构建体：利用烟草花叶病毒的35S启动子的过表达载体p35S::AhFH1，其载体骨架为植物过表达载体PHB，其含有所述花生开花习性基因AhFH1相关核苷酸序列，该过表达载体构建所需的引物对为OE-FH1-F和OE-FH1-R，其序列如SEQ ID NO：17-18所示；利用该引物对在交替开花花生的cDNA中或含有该基因完整编码框的质粒中扩增获得基因AhFH1，将扩增产物通过酶切连接或重组连接入过表达载体PHB(如图7)或其它植物过表达载体，构建成为一种过表达转基因载体p35S::AhFH1(如图7A)。将该过表达载体转化入连续开花型花生可以提高花生的分枝数目，进而影响与其相关的其他性状。
本发明还提供一种互补表达重组构建体：以上述构建的过表达转基因载体p35S::AhFH1为基础，利用基因AhFH1的自身启动子构建互补表达转基因载体：pFH1::AhFH1，其含有所述花生开花习性基因AhFH1相关核苷酸序列。所述互补表达载体构建所需的引物对为FH1pro-F/R，其序列如SEQ ID NO：19-20所示，上游引物FH1pro-F的EcoR I酶切位点为“ gaattc”，下游引物FH1pro-R的Pst I酶切位点为“ ctgcag”。利用该引物对交替开花花生品种的DNA进行启动子克隆扩增，将连入T载体的扩增产物或扩增产物直接通过EcoR I和Pst I双酶切回收后的目的片段连接入利用同样酶切线性化的过表达转基因载体p35S::AhFH1回收的大片段，构建成为一种互补表达转基因载体pFH1::AhFH1(如图7B)。另外 互补表达载体的构建也可以利用适当的引物直接扩增交替开花品种中功能性AhFH1包含启动子和编码区在内的基因组全长连入适当的植物转基因载体，此处不再赘述。将该互补表达载体转化入连续开花型花生可以将其改变为交替开花型花生，提高分枝数目，进而影响与其相关的其他性状。
本发明还提供一种基因编辑载体构建体，其含有本发明所述的AhFH1或等位基因Ahfh1的部分核苷酸序列，所述载体是基因编辑载体KO-AhFH1。用于构建基因AhFH1基因编辑的目标靶点的序列优选有两种，分别为：sgRNA1和sgRNA2，其序列如SEQ ID NO：21-22所示；将该两个片段分别连接进入CRISPR/Cas9载体BGK041(图8)的sgRNA区分别构建成针对目标基因AhFH1的基因编辑敲除载体KO-AhFH1。将该基因编辑载体转化入交替开花型花生品种可以将其AhFH1基因实现基因编辑而改变，进而挑选出去功能性编辑后代，而实现交替开花型花生转变为连续开花型，降低其分枝数目、增加其开花数目、荚果数目以及其相关的其他性状。所述sgRNA1和sgRNA2为优选靶标序列，根据不同的CRISPR/Cas9载体系统或编辑效率，靶标序列可以不同。
本发明所述的花生开花习性基因AhFH1及其等位变异直接来源于花生，也可以来源于大豆、油菜、棉花、水稻、玉米、小麦等农作物的相似度足够高的同源基因。
本发明还提供改良花生开花习性性状的方法，该方法包括制备含有上述中AhFH1或等位基因Ahfh1相关核苷酸序列构建体的花生植株。
本发明具有以下有益效果：
本发明所提供的花生开花习性基因AhFH1的克隆与应用，具有以下有益效果：
(1)本发明提供的花生开花习性基因AhFH1及其等位变异，为探索花生开花习性基因AhFH1调控花生开花习性的分子机制和初步构建其参与开花分枝调控的分子网络，以及研究该基因功能在作物间的演化规律等提供重要的参考。
(2)本发明提供的花生开花习性基因AhFH1及其等位变异基因间的差异，可以开发为功能性分子标记，用于作物分子标记辅助选择育种，优选在改良花生的开花习性及与其相关的分枝数目、荚果数目、荚果集中程度、成熟一致性和荚果产量等性状中起到关键性的作用。
(3)本发明提供的花生开花习性基因AhFH1的基因序列和氨基酸或多肽或蛋白质在作物遗传改良中，优选在改良花生的开花习性及与其相关的分枝数目、荚果数目、荚果集中程度、成熟一致性和荚果产量等性状中起到关键性的作用。
(4)本发明提供的花生开花习性基因AhFH1的过表达载体、互补表达载体和基因编辑载体及含有所述载体的植株，优选在改良花生的开花习性及与其相关的分枝数目、荚果数目、荚果集中程度、成熟一致性和荚果产量等性状中起到关键性的作用。
附图说明：
图1为本发明涉及的花生开花习性模式图，A为连续开花型，B为交替开花型。
图2为本发明涉及的花生开花习性基因AhFH1的图位克隆过程图。
图3为本发明涉及的花生开花习性基因AhFH1在花生代表性品种基因组水平基因全长的克隆电泳图(引物对FH1g-F/R)。
图4为本发明涉及的花生开花习性基因AhFH1的cDNA克隆电泳图(引物对FH1cd-F/R)。
图5为本发明涉及的鉴别AhFH1和Ahfh1-1两种等位变异的功能性分子标记InDel-1492bp的电泳图(引物对InDel-1492bp-F/R)。
图6为本发明涉及的花生开花习性基因AhFH1的两种启动子在基因组中克隆电泳图(引物对FH1p-F/R)。
图7为本发明涉及的构建体p35S::AhFH1和pFH1::AhFH1的结构图。
图8为本发明涉及的基因编辑构建体KO-AhFH1的结构图。
具体实施方式：
下面通过实施例结合附图对本发明技术做进一步说明。范例中所用的DNA提取、RNA提取、RNA反转录为cDNA、PCR扩增、酶切连接等分子生物学手段不做特殊说明均为常规分子生物学实验手段，可以查阅《分子生物学实验指南》。
实施例1：花生开花习性基因AhFH1的图位克隆
(1)花生品种材料和杂交群体的构建：268份花生品种种质资源由青岛农业大学花生中心保藏。从中挑选交替开花型栽培花生Florunner与连续开花型栽培花生平度9616杂交，种植杂交种F 1，自交获得F 2分离群体，连续多代株系自 交繁育，最终形成F 6代重组自交系PF-F 6。交替开花型栽培花生小红毛与连续开花型栽培花生河南南阳杂交，种植杂交种F 1，自交获得F 2分离群体，连续多代株系自交繁育，最终形成F 7代重组自交系HN-F 7。
(2)植物DNA的提取：采用改良的CTAB法提取植物DNA。
(3)植物RNA的提取及反转录：采用Takara的RNA Extraction Kit提取RNA，反转录为cDNA采用的是Takara的PrimeScript TM RT reagent Kit。
(4)利用Advanced-BSR-Seq(Advanced Bulked Segregation RNA sequencing)的方法初定位花生开花习性基因AhFH1(引用专利“一种同时定位两个性状相关基因的方法CN110675915A”)：
对平度9616和Florunner以及60个个体(30个交替开花型和30个连续开花型)分别进行转录组测序，获得62个样品的转录组测序数据。通过转录组数据表与栽培品种Tifrunner参考基因组序列比对筛选SNP结果，最终得到高质量的可信的SNP位点12,421个。对高质量的SNP进行交替开花池和连续开花池间的△SNP-index分析，将开花习性基因初定位在第12号染色体末端，即第12号染色体117 682 534bp到119 846 824bp之间，总长度约为2.16M(Tifrunner参考基因组，第一版)。
(5)花生开花习性基因AhFH1的精细定位和候选基因预测：
利用连续开花花生品种平度9616和交替开花花生品种Florunner构建的重组自交系的全群体，采用连锁定位的方式将控制花生开花习性的基因定位在第12染色体的末端InDel标记P-21和P-29之间(长度约0.89Mb)。进一步，针对连锁定位验证的标记P-21和P-29间筛选获得的25个重组个体(群体由445个株系构成)，继续用内部的InDel标记进行基因型鉴定，结合表型分析将目标位点缩小在InDel标记P-21与SR-4之间的446kb区间内。根据亲本基因组重测序数据在该区间内设计了多个测序片段进行双亲测序，获取了亲本间的2个SNP标记，对内部的9个重组个体进行测序和表型对照，最终将这一位点缩小在P-21与SNP-6之间的387KB的区间内(如图2，标记的引物见表1)。在这个387KB区间里，参考基因组共预测注释有约44个基因，通过生物信息学分析，将区间内一个PEBP/CET基因家族(成花素FT基因家族)的基因Arahy.BBG51B初步选定为花生开花习性基因AhFH1的候选基因。
表1 精细定位的标记的引物信息
Figure PCTCN2020118264-appb-000001
(6)花生开花习性基因AhFH1的克隆及关联验证：
通过精细定位将Arahy.BBG51B初步选定为花生开花习性基因AhFH1的候选基因。对这一候选基因在参考基因组Tifrunner(交替开花型)和狮头企(连续开花型)间进行序列比对发现：在狮头企(连续开花型)的候选基因的启动子区存在一个214bp的插入，而狮头企的编码区参考序列不完整。根据Tifrunner的参考序列设计针对基因AhFH1基因组水平的克隆引物FH1g-F/R(SEQ ID NO:4-5)，使用该引物对以交替开花花生的基因组DNA为模板，通过PCR扩增克隆所述候选基因的基因组完整序列(如图3)。根据参考序列设计了从cDNA中克隆基因AhFH1的引物对FH1cd-F/R，其序列如SEQ ID NO：6-7所示，使用该引物对以交替开花花生的侧枝茎端或叶片组织的cDNA为模板，通过PCR扩增克隆所述候选基因的完整编码框(如图4)。本实施例涉及的代表性的栽培品种为小红毛、河南南阳、Florunner、平度9616、四粒红、鲁花11、马渐渐103、龙花生559、Tifrunner和狮头企。由于在基因全长的扩增中(引物对FH1g-F/R)，确实存在代表性的栽培品种未克隆出基因组水平的基因全长，因此对候选基因下游继续设计跨度更大的引物对，其中引物对可以扩增出较小的片段对应的等位变异Ahfh1-1(SEQ ID NO：8)，对扩增片段进行测序发现其存在一个1492bp的缺失，将这一引物对命名为InDel-1492bp-F/R，其序列如SEQ ID NO：9-10所示，可以直接鉴别AhFH1和Ahfh1-1两种等位变异，对应等位变异AhFH1扩增产物为2556bp，对应等位变异Ahfh1-1的扩增产物为1064bp，可以通过琼脂糖电泳 检测其差异(如图5)。利用该分子标记可以在等位差异为AhFH1和Ahfh1-1的品种间杂交育种中，进行开花习性等位变异的分子标记辅助选择，或在种质资源中进行等位变异AhFH1和Ahfh1-1的鉴别。
根据参考序列设计针对启动子的克隆引物FH1p-F/R(SEQ ID NO:15-16)，在代表品种中进行候选基因AhFH1启动子的克隆，并对目的条带进行测序比较分析。通过克隆和测序比较分析发现候选基因AhFH1的启动子序列有两种主要类型，分别来自Tifrunner和狮头企(SEQ ID NO:13-14)，但在种质中的克隆条带有三种类型，除了单一的Tifrunner型短带和狮头企型的长带，还存在长短两条带的双带型(如图6)，结合后期研究结果分析发现双带型为异源四倍体花生A和B两套亚基因中的两种启动子类型均存在。
通过对启动子区和基因末端的克隆发现，亲本小红毛(交替开花型)的A和B基因组上的序列是完整并且完全一致的，不存在启动子的插入和基因末端的缺失。平度9616(连续开花型)和河南南阳(连续开花型)的A基因组和B基因组上该基因的序列也完全一致，但其启动子存在214bp的插入和基因末端存在1492bp的缺失。然而亲本Florunner(交替开花型)在启动子和基因末端扩增中均出现两个条带，经测序分析发现两个条带都是目的条带，分析为A基因组和B基因组上存在等位差异，在A基因组上存在启动子的插入和基因末端的缺失，在B基因组上不存在启动子的插入和基因末端的缺失。由此可见：平度9616和Florunner之间B基因组上存在差异。这一结果，也验证了小红毛和河南南阳构建的RIL群体开花习性由两个位点控制，同时也与平度9616和Florunner构建的RIL群体的开花习性由一个位点控制相吻合。通过对代表性栽培花生品种进行测序比对发现的启动子区(214bp)的插入和基因末端(1492bp)的缺失可能影响花生的连续开花型和交替开花型，因而将花生开花习性候选基因上发现的启动子区(214bp)的插入和基因末端(1492bp)的缺失开发为InDel标记，分别定义为FH1p-F/R和InDel-1492bp。在开花习性丰富的268份品种种质资源中进行关联验证发现除了启动子区(214bp)的插入与表型的关联度不高，基因末端1492bp缺失型种质均为连续开花型，但基因末端不缺失的种质也有多个是连续开花型，针对非缺失但连续开花的种质进行了基因AhFH1的cDNA水平的基因编码区的克隆和测序，发现该类种质的AhFH1基因的第4外显子上编码的cDNA 的+335bp位置的碱基C缺失，导致提前形成终止子，翻译的蛋白缺失63个氨基酸，从而影响花生的开花习性，即为等位变异Ahfh1-2(SEQ ID NO:11)。
结合启动子区的插入与否(214bp)和基因末端的缺失与否(1492bp)以及第4外显子上编码的cDNA的+335bp位置的碱基C缺失，这三个位点对268份种质资源进行关联分析发现268份种质资源表型和AhFH1的去功能与否的吻合度达到了百分之百，完整功能的AhFH1对应交替开花，去功能性Ahfh1-1或Ahfh1-2对应连续开花，A和B亚基因组杂合对应交替开花。至此，我们将这一候选基因Arahy.BBG51B确定为花生开花习性基因AhFH1(Flowering Habit 1 of Arachis hypogaea L.)，基因AhFH1存在一种完整功能性的等位变异AhFH1和至少两种去功能性等位变异Ahfh1-1和Ahfh1-2。
通过分析交替开花型测序品种Tifrunner的参考序列，发现其不同套染色体的同源染色体A02和B02在这一候选基因的上下游约500kb的范围内几乎是完全相同的，可能是A、B亚基因组间易位导致的，同时对A02的基因组注释的Arahy.DYRS20与Arahy.BBG51B完全相同，故本实施例所述AhFH1包含有两个位点：A02染色体的Arahy.DYRS20(将其命名为AhFH1A)与B02染色体的Arahy.BBG51B(将其命名为AhFH1B)。结合基因的克隆和关联验证分析表明：花生开花习性基因AhFH1在A和B两套亚基因组构成的异源四倍体栽培花生中理论是存在四种基因型：AhFH1A/AhFH1B、Ahfh1a/Ahfh1b、Ahfh1a/AhFH1B和AhFH1A/Ahfh1b，而通常在同一个品种中，A完全等于B，a完全等于b，所以实际可以简单地将其分为三种基因型：AhFH1/AhFH1、Ahfh1/Ahfh1和AhFH1/Ahfh1，其中AhFH1/AhFH1和AhFH1/Ahfh1为交替开花基因型，只有Ahfh1/Ahfh1为连续开花型。对于交替型的两个亲本杂交分离出现连续开花型，理论上是Ahfh1a/AhFH1B和AhFH1A/Ahfh1b两种基因型品种杂交，后代出现重组型Ahfh1a/Ahfh1b，对应的表型为连续开花。
实施例2：花生开花习性基因AhFH1的过表达转基因应用
本实施例利用烟草花叶病毒的35S作为启动子构建过表达转基因载体p35S::AhFH1，通过花粉管导入法将花生开花习性基因AhFH1的mRNA在连续开花型品种(花育23)中进行过表达，具体步骤包括：用Sac I和Xba I双酶切将过表达载体PHG上的GFP切下，以T质粒为模板利用同源重组的引物OE-AhFH1-F 和OE-AhFH1-R，其序列如SEQ ID NO：17-18所示，扩增目的片段，胶回收T质粒扩增的目的片段和过表达载体PHB骨架片段，纯化后用同源重组的方法进行连接，将连接产物热激转化大肠感菌DH5a感受态细胞，涂布卡那霉素抗性的LB平板，挑取单克隆通过PCR检测，将阳性克隆送去青岛擎科梓熙生物技术有限公司测序，选择测序正确的菌株摇菌，提取带有目的片段的质粒，即为AhFH1过表达转基因载体：p35S::AhFH1，其结构如图7A所示。将AhFH1过表达载体转化农杆菌GV3101感受态细胞，涂布卡那霉素和利福平双抗的YEB平板，挑取单克隆后通过PCR检测阳性后作转基因菌株备用。将该过表达转基因载体转化入连续开花型花生可以提高分枝数目，进而影响与其相关的其他性状。
实施例3：花生开花习性基因AhFH1自身启动子的互补表达应用
该互补表达转基因载体构建所需的引物对为FH1pro-F和FH1pro-R，其序列如SEQ ID NO：19-20所示，利用该引物对在交替开花花生的DNA模板中进行克隆扩增，将连入T载体的扩增产物或扩增产物直接通过EcoR I和Pst I双酶切回收后的目的片段连接入利用同样酶切线性化的过表达转基因载体p35S::AhFH1，构建成为一种互补表达转基因载体pFH1::AhFH1(如图7B)。
FH1pro-F:5`-CG GAATTCACGAAATCTCAACTTGTTTACGT-3`(SEQ ID NO：18)
FH1pro-R:5`-AA CTGCAGTGTTAAAGAGAATGAAAGAGAA-3`(SEQ ID NO：19)；(FH1pro引物：上游AhFH1pro-F的EcoR I酶切位点为“GAATTC”，下游FH1pro-R的Pst I酶切位点为“CTGCAG”)。
互补表达转基因载体的构建也可以利用适当的引物直接扩增交替开花品种中功能性AhFH1包含启动子和编码区在内的基因组全长连入适当的植物转基因载体，此处不再赘述。
以花生开花习性基因AhFH1的自身启动子作为启动子构建过表达载体，通过花粉管导入法将花生开花习性基因AhFH1的mRNA在连续开花型品种(花育23)中进行过表达，具体步骤包括：以构建的35S启动子的过表达载体p35S::AhFH1为基础进行自身启动子表达载体构建；过表达载体p35S::AhFH1利用EcoR I和Pst I双酶切，切除35S启动子序列，回收过表达载体p35S::AhFH1的大片段约12kbp；利用引物对为FH1pro-F/R，其序列如SEQ ID NO：19-20所 示，克隆栽培花生小红毛的AhFH1基因的启动子，并与T载体进行连接和转化，进行测序提取质粒，再利用EcoR I和Pst I对提取的质粒进行双酶切，回收目标片段，然后利用T4连接酶将回收的过表达载体p35S::AhFH1大片段和T载体扩增出来的EcoR I和Pst I双酶切回收后的目的片段进行连接并转化大肠杆菌，提质粒酶切检测和测序验证正确性，即为AhFH1自身启动子的互补表达载体pFH1::AhFH1，其结构如图7B所示。将AhFH1自身启动子的过表达载体转化农杆菌GV3101感受态细胞，涂布卡那霉素和利福平双抗的YEB平板，挑取单克隆后通过PCR检测阳性后作转基因菌株备用。将该互补表达载体转化入连续开花型花生可以将其改变为交替开花型花生，提高分枝数目，进而影响与其相关的其他性状。
实施例4：花生开花习性基因AhFH1的基因编辑的敲除应用
本实施例利用CRISPR/Cas9体系进行基因编辑敲除，具体操作步骤包括：在线设计并生成sgRNA靶点序列(http://www.biogle.cn/index/excrispr)，选择两个得分最高的靶位点sgRNA1和sgRNA2(SEQ ID NO：21-22)，将生成的sgRNA序列交由青岛擎科梓熙生物技术有限公司合成互补的两条单链Oligo，将合成的Oligo加水溶解至10μM，在200ul PCR管中加入18μl Buffer Anneal、1μl Up Oligo和1μl Low Oligo混合后，95℃加热3分钟，然后以约0.2℃/秒缓慢降至20℃，制备Oligo二聚体(具体参照百格载体说明书)；将Oligo二聚体通过连接酶连接至线性化的CRISPR/Cas9载体即为KO-AhFH1载体；在200ul PCR管中加入2ul KO-AhFH1载体、1ul Oligo二聚体、1μl Enzyme Mix和16ul ddH 2O，混匀后室温(20℃)反应1小时，将连接产物热激法分别转化大肠感菌DH5a感受态细胞，涂布卡那霉素抗性的LB平板，挑取单克隆，通过PCR检测，将阳性克隆送去青岛擎科梓熙生物技术有限公司测序，选择测序正确的菌株摇菌，提取质粒，即为AhFH1敲除质粒：KO-AhFH1-1/2。将AhFH1基因敲除质粒KO-AhFH1-1/2转化农杆菌感受态细胞，涂布卡那霉素和利福平双抗的YEB平板，挑取单克隆后通过PCR检测阳性，选择阳性克隆转化交替开花型花生(如小红毛或209小花生)；所述CRISPR/Cas9载体选用BGK041(如图8)，该载体采用大豆U6启动子驱动sgRNA序列，能够高效的用于双子叶植物，采用加强型CaMV 35S启动子高效表达Cas9蛋白。将该基因编辑载体转化入交替开花型花生品种可以将 其AhFH1基因实现基因编辑而改变，进而挑选出去功能性编辑后代，而实现交替开花型花生转变为连续开花型，降低其分枝数目、增加其开花数目、荚果数目以及其相关的其他性状。
基因编辑所用的CRISPR/Cas9载体BGK041骨架购买自百格基因公司(http://www.biogle.cn/index/excrispr)，仅用于范例说明，也可以采用其它植物CRISPR/Cas9基因编辑载体或其他单碱基编辑载体。

Claims (6)

  1. 一种花生开花习性基因AhFH1及其等位变异在作物遗传改良中的应用，其特征在于，优选在改良花生的开花习性及与其相关的分枝数目、荚果数目、荚果集中程度、成熟一致性和荚果产量等性状中的应用；所述花生开花习性基因AhFH1的核苷酸序列如SEQ ID NO:1所示，其编码的cDNA的核苷酸序列如SEQ ID NO:2所示，其编码的蛋白质的氨基酸序列如SEQ ID NO:3所示；所述基因AhFH1的启动子序列有两种主要类型，分别来自Tifrunner和狮头企，序列分别如SEQ ID NO:13-14所示；所述花生开花习性基因AhFH1的等位变异包括去功能性等位变异Ahfh1-1和Ahfh1-2，所述去功能性等位变异Ahfh1-1，其核苷酸序列如SEQ ID NO:8所示，其基因组水平的基因末端存在一个以ATG为起始的+1872bp至+3273bp位置的包含最后一个外显子及3`UTR的1492bp的缺失，其对应花生的开花习性为连续开花型；所述去功能性等位变异Ahfh1-2，其核苷酸序列如SEQ ID NO:11所示，其编码的cDNA序列如SEQ ID NO:12所示，其编码的cDNA的+335bp位置的碱基C缺失，导致cDNA翻译框移码提前形成终止子，翻译的蛋白不完整，其对应花生的开花习性为连续开花型。
  2. 花生开花习性基因AhFH1的克隆引物，其特征在于，具体包括：基因AhFH1在基因组水平的克隆引物对为FH1g-F/R，其核苷酸序列如SEQ ID NO:4和SEQ ID NO:5所示；基因AhFH1在cDNA水平的克隆引物对为FH1cd-F/R，其核苷酸序列如SEQ ID NO:6和SEQ ID NO:7所示；基因AhFH1启动子的克隆引物对为FH1p-F/R，其核苷酸序列如SEQ ID NO:15和SEQ ID NO:16所示，引物对FH1p-F/R同时可以用于对花生开花习性基因AhFH1启动子的两种不同类型的鉴别。
  3. 一种区分花生开花习性基因完整功能型AhFH1和去功能性Ahfh1-1的功能性分子标记InDel-1492bp，其特征在于，其对应的引物对InDel-1492bp-F/R的核苷酸序列分别如SEQ ID NO:9和SEQ ID NO:10所示，结合测序确认AhFH1序列无变异的情况下，标记InDel-1492bp可以通过PCR和电泳检测直接判断两种等位变异。
  4. 一种含有花生开花习性基因AhFH1的过表达转基因载体构建方法及应用，其特征在于，利用烟草花叶病毒的35S启动子构建过表达转基因载体p35S::AhFH1，其载体骨架为植物过表达载体PHB，其含有所述花生开花习性基因AhFH1相关核苷酸序列，所述过表达载体构建所需的引物对为OE-FH1-F和OE-FH1-R，其序列如SEQ ID NO：17-18所示；利用该引物对在交替开花花生的cDNA中或含有该基因完整编码框的质粒中扩增获得基因AhFH1，将扩增产物通过酶切连接或重组连接入过表达载体PHB或其它植物过表达载体，构建成为一种过表达转基因载体p35S::AhFH1；将该过表达载体转化入连续开花型花生可以提高花生的分枝数目， 进而影响与其相关的其他性状。
  5. 一种含有所述花生开花习性基因AhFH1相关核苷酸序列的互补表达转基因载体构建方法及应用，其特征在于，过表达转基因载体p35S::AhFH1为基础，利用基因AhFH1的自身启动子构建互补表达载体：pFH1::AhFH1，其含有所述花生开花习性基因AhFH1相关核苷酸序列；所述互补表达载体构建所需的引物对为FH1pro-F/R，其序列如SEQ ID NO：19-20所示，上游引物FH1pro-F的EcoR I酶切位点为“ gaattc”，下游引物FH1pro-R的Pst I酶切位点为“ ctgcag”。利用该引物对交替开花花生品种的DNA进行启动子克隆扩增，将连入T载体的扩增产物或扩增产物直接通过EcoR I和Pst I双酶切回收后的目的片段连接入利用同样酶切线性化的过表达转基因载体p35S::AhFH1回收的大片段，构建成为一种互补表达转基因载体pFH1::AhFH1，将该互补表达载体转化入连续开花型花生可以将其改变为交替开花型花生，提高分枝数目，进而影响与其相关的其他性状。
  6. 一种含有所述基因AhFH1或等位基因Ahfh1的基因编辑载体构建方法及应用，其特征在于，所述载体是基因编辑载体KO-AhFH1；用于构建基因AhFH1基因编辑的目标靶点的序列优选有两种，分别为：sgRNA1和sgRNA2，其序列如SEQ ID NO：21-22所示；将该两个片段分别连接进入CRISPR/Cas9载体BGK041的sgRNA区分别构建成针对目标基因AhFH1的基因编辑敲除载体KO-AhFH1；将该基因编辑载体转化入交替开花型花生品种可以将其AhFH1基因实现基因编辑而改变，进而挑选出去功能性编辑后代，实现交替开花型花生转变为连续开花型，降低其分枝数目、增加其开花数目、荚果数目以及其相关的其他性状；所述sgRNA1和sgRNA2为优选靶标序列，根据不同的CRISPR/Cas9载体系统或编辑效率，靶标序列可以不同。
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