CN114854786B - A method for improving the induction rate of maize haploid induction lines by genetically engineering CENH3 protein - Google Patents

Abstract

The invention discloses a method for improving the induction rate of a corn haploid induction line by genetically engineering CENH3 protein, and particularly relates to the field of genetic engineering. The method comprises the steps of replacing the sequence of the N end in a corn CENH3 gene with the N end sequence of a corn H3 gene to obtain a preliminary modified gene 1, cloning the gene 1 into a plant over-expression vector pCAMBIA3301-RFP which takes Ubiquitin (UBI) as a promoter and contains a macromolecular Red Fluorescent Protein (RFP) tag, constructing a vector UBI for over-expressing a centromere specific fusion protein, namely M-tailswap-RFP, and transforming the over-expression vector into a corn receptor to obtain a positive transformant strain LH244 ^{M‑tailswap‑RFP}; hybridizing the positive strain LH244 ^{M‑tailswap‑RFP} with the receptor parent CAU5 for 1 generation, then backcrossing with the CAU5 as the recurrent parent for 2 generation, and selfing for at least 4 generation. The breeding method can obviously improve the induction rate of the corn haploid induction system, and haploid induction is carried out by using the corn haploid induction system containing fluorescent markers, so that the efficiency of identifying haploids in the field can be improved, and the identification process is simplified.

Description

A method for improving the induction rate of maize haploid induction lines by genetically engineering CENH3 protein

Technical Field

The invention relates to the field of genetic engineering, and in particular to a method for improving the induction rate of maize haploid induction lines by genetically engineering CENH3 protein.

Background Art

Corn is a model crop that utilizes hybrid vigor. More than 97% of corn planting area in my country uses hybrids. The most critical link in the utilization of hybrid vigor is the selection of pure lines. There are two commonly used methods: the first is the pedigree method, which is to obtain pure lines through continuous multi-band selfing or backcrossing, which usually takes 5-7 years; the other is the double haploid (DH) breeding technology, which uses the haploid induced by the haploid induction line to double and become a homozygous diploid, and pure lines can be obtained within 1-2 years. DH breeding plays an increasingly important role in the commercial breeding process of corn, and has become the core technology of modern corn breeding together with molecular breeding technology and transgenic technology.

Haploid refers to an individual with the same number of gamete chromosomes in its cells. Haploids have only one set of chromosomes, and both recessive and dominant genes can be manifested in the present generation. Therefore, excellent traits can be screened in the early generation, and undesirable traits can be eliminated in time, which is conducive to the rapid aggregation of favorable alleles such as yield and resistance. After the haploid is doubled, homozygous individuals can be obtained directly without gene separation, and the time to obtain pure lines is shorter than that of conventional breeding pedigree methods. In addition, since there is no gene interaction in haploid breeding, molecular marker-assisted selection can improve the efficiency and accuracy of breeding. In addition, the DH system can also be used to quickly generate mapping populations, chromosome substitution systems, reverse breeding parents, and apomixis engineering, etc.

At present, in the actual application of corn DH breeding, haploid offspring are mainly obtained by inducing maternal haploids through haploid induction lines derived from corn Stock6 germplasm. The Stock6 corn haploid induction line was first reported in 1959, with a maternal haploid induction rate of 2.3%-3.2%. In the following decades, corn breeders around the world have continuously improved the Stock6 induction line through hybridization or backcrossing, significantly improving the induction rate of corn haploid induction lines.

In addition to the haploid induction line breeding method based on Stock6 germplasm, CENH3-mediated haploid induction technology is another haploid acquisition method that is most likely to be used in commercial breeding programs in the future. The CENH3 gene encodes a centromere-specific histone, which is a variant of nucleosomal histone H3 and plays an important role in the positioning of centromeres on chromosomes. Current studies have shown that by using genetic engineering methods, by knocking out or mutating the CENH3 gene in plants such as corn, rice, and Arabidopsis, the gametophyte or sporophyte of the plant can be made to have the ability to induce the production of haploid offspring, thereby creating a haploid induction line.

However, the above two methods for breeding or creating haploid induction lines still have obvious defects. The method of breeding maize haploid induction lines by improving Stock6 germplasm currently has the following main disadvantages: 1. Even after years of backcross breeding, the rate of improvement of the induction rate of haploid induction lines is still slow. 2. Due to the limitation of germplasm resources, the improvement of agronomic traits of haploid induction lines and the improvement of haploid induction rate cannot be fully taken into account, resulting in materials with higher induction rates, whose agronomic traits may be poor and cannot be used for commercial breeding applications.

The method of creating a haploid induced corn line by genetically engineering the CENH3 gene currently has the following disadvantages: 1. The induction rate of the haploid induced line is still low, and the haploid induction rate of each plant in the created induced line population is unstable. 2. Since most conventional corn lines do not carry color marker genes in their genetic background, it is difficult to identify the haploid induced by the induced lines created using them.

Summary of the invention

To this end, the present invention provides a method for improving the induction rate of maize haploid induction lines by genetically engineering CENH3 protein, so as to solve the problems of slow improvement of existing maize haploid induction rate, low efficiency of haploid identification, and poor agronomic traits of induced lines.

In order to achieve the above object, the present invention provides the following technical solutions:

According to the present invention, a method for improving the induction rate of maize haploid induction lines by genetically engineering CENH3 protein is provided, the method comprising the following steps:

Step 1: Replace the N-terminal sequence of the corn CENH3 gene with the N-terminal sequence of the corn H3 gene to obtain the preliminary transformed gene 1, and then clone the gene 1 into the plant overexpression vector pCAMBIA3301-RFP with Ubiquitin (UBI) as the promoter and containing a large molecular weight tag to construct a vector UBI:M-tailswap-RFP that overexpresses the centromere-specific fusion protein, and transform the overexpression vector into the corn recipient to obtain the positive transformation strain LH244 ^{M-tailswap-RFP} ;

Step 2: hybridize the positive strain with the recipient parent corn CAU5 for one generation, then backcross for two generations with CAU5 as the recurrent parent, and then self-pollinate for at least four generations to finally form a new haploid induced line with a high induction rate.

Furthermore, in step 1, the high molecular weight tag is RFP fluorescent protein.

Furthermore, in step 1, the centromere-specific fusion protein is specifically a fusion expression protein in which the N-terminal amino acid sequence of the corn centromere-specific histone CENH3 is replaced with the N-terminal amino acid sequence of the corn nucleosome histone H3, and a high molecular weight fluorescent label RFP is connected to the C-terminus of the CENH3 protein.

Furthermore, in the step 1, the transformation is carried out by the Agrobacterium embryo immersion method.

Furthermore, in step 1, the corn recipient strain used is the LH244 strain.

Furthermore, in step 2, the recipient parent corn is the corn haploid induction line CAU5 derived from Stock6.

The purpose of the cultivation method of the present invention for selecting the C-terminus of corn CENH3 to splice the N-terminus of corn H3 gene and connect the fluorescent protein is that the chimeric gene formed by splicing H3 and CENH3 is still specifically expressed in the centromere, and it is introduced into corn, so that the fluorescent marker can be stably expressed in the cell nucleus. In addition, since the CENH3 chimeric protein spliced with the N-terminal sequence of the H3 protein may not have the function completely consistent with the wild-type CENH3 protein, and its C-terminus is fused with an exogenous macromolecular fluorescent protein label RFP, when it is integrated into the centromere, it has a certain impact on the centromere function. When competing with the natural CENH3 protein from the mother, it may be due to the lag in the time node of integration into the centromere, or the efficiency of competing for spindle pulling is not as good as the natural CENH3 protein in the normal mother, so the chromosome containing the spliced CENH3 fusion protein (M-tailswap-RFP) is more likely to lag in mitosis and be lost.

The present invention has the following advantages:

The breeding method of the present invention significantly improves the induction rate of the corn haploid induction line. The corn haploid induction line with fluorescent marker can not only be used as a haploid induction line to produce haploids in offspring, but also can stably express fluorescent fusion protein, which greatly simplifies the identification process; improves the efficiency of identifying haploids in the field and simplifies the identification process.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for the embodiments or the description of the prior art are briefly introduced below. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

FIG1 is a schematic diagram of the construction of a vector (UBI:M-tailswap-RFP) provided by the present invention, in which the N-terminus of a maize CENH3 protein is replaced with the N-terminus of a maize H3 protein, and a red fluorescent protein tag (RFP) is connected to the C-terminus of the chimeric CENH3 protein;

FIG2 is the identification result of the transgenic line using the primers for amplifying the sequence after splicing the N-terminus of H3 and the C-terminus of CENH3 provided by the present invention; wherein, lanes 1 and 2 are biological replicates of DNA of two positive plants of LH244 ^{M-tailswap-RFP} , and lane 3 is a marker, and the brightest band points to 750bp;

FIG3 is a comparison of CENH3 gene expression levels between LH244 ^{M-tailswap-RFP} positive overexpression strain and control strain using real-time fluorescence quantitative PCR (qRT-PCR) provided by the present invention;

FIG4 is a flow chart of a group combination provided by the present invention;

FIG5 is a data comparison diagram of haploid induction rates of various generations during the process of introducing the UBI:M-tailswap-RFP overexpression vector into the maize CAU5 induction line through backcross breeding provided by the present invention;

FIG6 is a comparison diagram of the pollen viability staining of CAU5 provided by the present invention and the newly bred novel induction line CAU5 ^{M-tailswap-RFP} ;

FIG7 is a comparison chart of the proportions of pollen of five vigor levels of CAU5 provided by the present invention and the newly bred novel induction line CAU5 ^{M-tailswap-RFP} ;

FIG8 is a comparison diagram of plant photos of the novel fluorescence induction lines CAU5 ^{M-tailswap-RFP} and CAU5 bred by the present invention.

DETAILED DESCRIPTION

The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example Method for improving the induction rate of maize haploid induction line by modifying maize CENH3 gene

illustrate:

The nucleotide sequence of the maize CENH3 gene is shown in the sequence list <210>1, NO.1 seq, 2 Ambystoma lateralex Ambystoma jeffersonianum-1; the amino acid sequence deduced from the nucleotide sequence of the maize CENH3 gene is shown in the sequence list <210>2, NO.2 seq, 2 Ambystoma laterale x Ambystoma jeffersonianum-2.

The nucleotide sequence of the maize HISTONE3.2 (H3) gene is shown in the sequence list <210>3, NO.3 seq, 2 Ambystoma laterale x Ambystoma jeffersonianum-3; the nucleotide sequence of the maize CENH3 spliced maize H3 gene is shown in the sequence list <210>4, NO.4 seq, 2 Ambystoma laterale x Ambystoma jeffersonianum-4.

The nucleotide sequence of corn CENH3 spliced corn H3 gene is shown in sequence list <210>5, NO.5 seq, 2 Ambystomalaterale x Ambystoma jeffersonianum-5; the amino acid sequence deduced from corn CENH3 spliced corn H3 gene is shown in sequence list <210>6, NO.6 seq, 2 Ambystoma laterale x Ambystoma jeffersonianum-6.

The nucleotide sequence of the reporter gene RFP is shown in the sequence list <210>7, NO.7 seq, 2 Ambystoma lateralex Ambystoma jeffersonianum-7; the deduced amino acid sequence of the reporter gene RFP is shown in the sequence list <210>8, NO.8seq, 2 Ambystoma laterale x Ambystoma jeffersonianum-8.

Primers for amplifying the N-terminal sequence of H3 and the C-terminal sequence of CENH3:

F:5'ATGGCCCGCACGAAGCAGA3' (see sequence listing <210>9, NO.9 seq, 2 Ambystomalaterale x Ambystoma jeffersonianum-9)

R:5'CCTCGGGGAAGGACAGCTTC3' (see sequence listing <210>10, NO.10 seq, 2 Ambystomalaterale x Ambystoma jeffersonianum-10)

ZmPLA1-primer sequence

F:5'ACGGAAGGAGTAAGAGGATGTTT3' (see sequence listing <210>11, NO.11 seq, 2 Ambystomalaterale x Ambystoma jeffersonianum-11)

R:5'CGGGTAGTCCTTCCCGTTCAC3' (see sequence listing <210>12, NO.12seq, 2 Ambystomalaterale x Ambystoma jeffersonianum-12)

Primers for Bar gene

F:5'GCAAAGTCTGCCGCCTTACAAC3' (see sequence listing <210>13, NO.13 seq, 2 Ambystomalaterale x Ambystoma jeffersonianum-13)

R:5'TGTTATCCGCTCACAATTCCACAC3' (see sequence listing <210>14, NO.14 seq, 2 Ambystomalaterale x Ambystoma jeffersonianum-14)

(I) Construction diagram and method of the vector for splicing the N-terminus of the corn H3 gene to the C-terminus of the CENH3 gene and then connecting to the red fluorescent protein (RFP)

The schematic diagram of constructing the vector of splicing the N-terminus of corn H3 with the C-terminus of corn CENH3 and then connecting to red fluorescent protein (RFP) is shown in FIG1 . The specific steps are as follows:

1. Extract total RNA from fresh leaves of maize inbred line B73 plants, and reverse transcribe the total RNA into cDNA;

2. According to the N-terminal sequence of the corn H3 gene, a specific primer is designed, a restriction site XbaI is added before the forward primer, and a restriction site SpeI is added after the reverse primer, and the cDNA is amplified by the specific primer by a PCR method to obtain a target fragment of the spliced H3 gene containing the restriction site; according to the C-terminal sequence of the corn CENH3 gene, a specific primer is designed, a restriction site SpeI is added before the forward primer, and a restriction site KpnI is added after the reverse primer, and the cDNA is amplified by the specific primer by a PCR method to obtain a target fragment of the spliced CENH3 gene containing the restriction site;

3. The H3 and CENH3 gene fragments were double-digested with restriction endonucleases XbaI/SpeI and SpeI/KpnI, respectively. The pCAMBIA3301-RFP vector was double-digested with the corresponding restriction endonucleases XbaI/KpnI, and the digestion products of each gene fragment and vector were subjected to agarose gel electrophoresis and recovered by gel cutting;

4. The H3 and CENH3 gene fragments and the pCAMBIA3301-RFP vector digestion recovery products were connected by T4 ligase, and then the connection products were transformed into Escherichia coli DH5α strains, and the positive single clones containing the connection products were screened by PCR technology;

5. Amplify the positive monoclonal colonies, extract the plasmids, and send them to a biological company for sequencing of the target fragments. By comparing the returned sequencing results, extract the plasmids from the bacterial solution containing the vector of the required correct target gene fragment for use in the next genetic transformation.

(II) Obtaining transgenic lines

The genetic transformation of corn was carried out by Agrobacterium embryo immersion method, and the corn recipient strain was LH244 strain. The specific operation process is as follows:

1. The vector plasmid constructed in (a) was transferred into the Agrobacterium EHA105 strain, and the positive monoclonal colonies containing the target vector UBI:M-tailswap-RFP were screened by PCR technology, and the positive Agrobacterium colonies were expanded.

2. Select corn ears of LH244 strain 10-12 days after self-pollination as materials for callus induction, separate immature embryos from kernels on corn ears in a clean bench, and temporarily store the collected immature embryos in a hypertonic culture medium for later use;

3. Cultivate the Agrobacterium culture containing the target vector UBI:M-tailswap-RFP until the OD value is equal to 0.8, collect the cells by centrifugation, and resuspend the cells with a resuspension solution containing acetosyringone. Soak the collected immature embryos in the Agrobacterium resuspension solution for 5 minutes, then transfer them to the co-cultivation medium and culture them in the dark at 25°C for 7 days;

4. Transfer the young embryos after the dark culture stage to the gradient screening medium containing resistance, transfer once every two weeks, for a total of 3 rounds of screening;

5. The young embryos that have been screened and formed callus tissue are transferred to differentiation medium and cultured for 2 weeks to obtain regenerated plants;

6. Harden the regenerated plants and then plant them in the field or greenhouse;

7. Take leaves from transformed plants in the field or greenhouse, extract DNA, and use PCR technology to screen positive transformed plants containing the target vector UBI:M-tailswap-RFP;

8. Extract total RNA from leaves of positively transformed plants, reverse transcribe it into cDNA, and use qRT-PCR technology, combined with the growth status of plants in the field, to screen plants with high expression of CENH3 chimeric gene and good growth status, and self-pollinate these selected plants during the flowering and pollination period to obtain homozygous transgenic offspring.

(III) Construction process of a new maize haploid induction line with high induction rate

After the transgenic lines identified as positive were self-pollinated for three generations and stabilized, the pollen of the induction line CAU5 was used to cross-pollinate with the transgenic positive plants and the recipient plants, respectively. The changing trends of the population induction rate and the field agronomic traits of their offspring F1, BC1F1, BC2F1, BC2F2, BC2F3 and BC2F4 generations were statistically analyzed.

In the breeding process of each generation, through the means of molecular marker-assisted selection, two pairs of primers (sequence amplification primers for the N-terminal of H3 and the C-terminal of CENH3 and ZmPLA1-primer sequence) were used to screen and retain the maize haploid induction gene locus ZmPLA1 and the maize H3 spliced CENH3 overexpression vector (UBI:M-tailswap-RFP) in the subsequent generations. The identification results are shown in Figure 2; at the same time, in the backcross generation, the strain with the highest haploid induction rate was selected for backcrossing in each generation, so as to finally obtain a new maize haploid induction line CAU5 ^{M-tailswap-RFP} ; the construction process is shown in Figure 4.

Experimental Example 1 Agarose gel electrophoresis of PCR-positive plants

1. The obtained transgenic lines were used to identify positive plants using primers for the Bar gene, and the target sequences in the positive plants were further determined using primers for amplifying the sequences after splicing the N-terminus of H3 and the C-terminus of CENH3.

2. The gene expression levels in the transgenic lines and the control group were identified. The identification method was:

Leaves of the transgenic line LH244 ^{M-tailswap-RFP} and the control group LH244 were collected in the field, and total RNA was extracted and reverse transcribed to obtain cDNA after preservation in liquid nitrogen. Primers were designed using Oligo7 software, and the ACTIN gene was used as the internal reference gene. The TB Green Fast qPCR Mix used in the experiment was from Takara (Cat. No.: RR430S), and the reaction system is shown in Table 1:

Table 1 Reaction system

Components Volume/μL TB Green Fast qPCR Mix 15 Primer-F 0.6 Primer-R 0.6 cDNA 2 _H2O 10.6 DyeII 1.2 Total volume 30

The fluorescence quantitative PCR instrument used was ABI7500 from Applied Biosystems, USA.

The reaction program was two-step PCR: preheating at 95°C for 30 seconds; the cycle program was: 95°C, 15 seconds, 60°C, 33 seconds (collecting fluorescence), and a total of 40 cycles were set.

Calculation method of relative expression: The 2 ^-ΔΔCT method was used to perform relative quantification of gene expression.

The expression level of the Actin gene in each sample was set to 1.0, and the relative expression level of the gene in the sample was subtracted and calculated. Each sample was repeated three times and the average value was taken.

3. Identification results

The results are shown in Figures 2 and 3. The positive transformation strain LH244 ^M - ^tailswap-RFP containing the UBI:M-tailswap-RFP vector can be amplified by vector-specific primers to obtain a nucleic acid electrophoresis band of about 750 bp in size as shown in Figure 2, and the expression level of the CENH3 gene in the positive transformation strain LH244 ^{M-tailswap-RFP} is significantly higher than that in the control strain LH244 as shown in Figure 3.

Experimental Example 2 Calculation method and comparison of haploid induction rate

1. Haploid identification method:

The method used in this experiment to identify haploids is through color marker selection, that is, haploids and diploids are judged by identifying the R-nj marker on the grains. We used this method to determine the induction rate of the new induction line and the control group. The specific experimental method is: when the new induction line CAU5 ^{M-tailswap-RFP} and the control group CAU5 ^{LH244-Intergressed} are used as male parents and hybridized with the conventional hybrid Zhengdan 958, purple endosperm purple embryos (diploid) and purple endosperm non-purple embryos (pseudohaploid) will be produced on the hybrid ears. Since color markers may be difficult to identify on some grains, the identified pseudohaploids will be planted in the next season, and the actual number of haploids will be screened through the field performance of haploids.

Calculation method of haploid induction rate:

The induction rate of a certain induction line = the number of haploid grains detected on the hybrid ear when the induction line is used as the male parent / the total number of grains on the hybrid ear.

2. The comparison of the induction rate between the new induction line and the control group is shown in Figure 5, where the CAU5 ^{LH244-introgressed} line is the transgenic recipient line LH244, which was backcrossed and bred using the same breeding strategy as the spliced CENH3 line. The results showed that the haploid induction rate of the new induction line CAU5 ^{M-tailswap-RFP} was significantly higher than that of the control induction line CAU5 ^{LH244-introgressed} starting from the BC2F1 generation; and after entering the selfing generation (BC2F2), the haploid induction rate of the new induction line CAU5 ^{M-tailswap-RFP} increased faster, and the population induction rate in the BC4F4 breeding generation reached about 16.3%, which was significantly increased by about 6.1% compared with the control group.

Experimental Example 3 Calculation method and comparison of pollen vitality

Calculation method and judgment standard of pollen vitality

Prepare a 1% TTC solution, take a mature pollen, gently squeeze out the pollen contents on a glass slide, drop a drop of TTC solution in the air, slowly cover with a coverslip, let it stand in a 37°C incubator for 5 minutes, and then observe it under a normal phase contrast microscope. The degree of pollen staining is divided into 5 levels: the first level is high vitality, which is manifested by a large area of pollen being dyed red; the second level is medium vitality, which is manifested by a large area of pink pollen with medium staining intensity; the third level of pollen is low vitality, which is manifested by a large area of pink and white pollen; the fourth level is pollen without vitality, which is manifested by all pure white pollen; and the fifth level is aborted grains, as shown in Figure 7. The proportions of the five types of pollen in the induced line CAU5 and the new induced line CAU5 ^{M -tailswap-RFP} were counted respectively. At least 10 pollen from different positions of each line were tested. Formula of TTC solution: Weigh 1.0g of triphenyltetrazolium chloride and dissolve it in 1000mL of pure water. Mix it by inverting it upside down and dispense it into 1mL centrifuge tubes. Wrap it in tin foil and keep it away from light. Be careful to keep it away from light when using it.

The comparison of pollen viability staining between CAU5 and the newly selected new induction line CAU5 M ^{-tailswap- RFP is shown in Figure 6; the comparison of the proportions of pollen of five vigor levels between CAU5 and the newly selected new induction line CAU5 M} -tailswap-RFP is shown in Figure 7. The results showed that the overall pollen vigor of the new induction line CAU5 ^{M-tailswap-RFP} was significantly lower than that of its donor parent CAU5, especially the proportion of low-vigorous pollen in the pollen of CAU5 ^{M-tailswap-RFP} increased significantly, indicating that the vigor of the induction line pollen may be correlated with the haploid induction ability.

Comparison of plant photos of the new fluorescent induction line bred in Experimental Example 4 and CAU5

The phenotype comparison of the selected new fluorescent induction line and CAU5 plant photos is shown in Figure 8. As can be seen from the figure, the new haploid induction line CAU5 ^{M-tailswap-RFP} bred by the method of the present invention has a significantly improved haploid induction rate and its agronomic traits are also significantly improved compared with its donor parent CAU5, with a higher plant height and a more developed male spike inflorescence.

In summary, the method of the present invention has high operational feasibility, mature technical solutions, and can significantly improve the induction rate of corn haploid induction lines within a shorter breeding cycle. The method of the present invention will effectively improve the working efficiency of current corn haploid breeding technology in production practice and has important guiding significance.

Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Sequence Listing

<110> Shenyang Agricultural University

<120> A method for improving the induction rate of maize haploid induction lines by genetically engineering CENH3 protein

<141> 2022-02-17

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 471

<212> DNA/RNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum-1

<400> 1

atggctcgaa ccaagcacca ggccgtgagg aagacggcgg agaagcccaa gaagaagctc 60

cagttcgagc gctcaggtgg tgcgagtacc tcggcgacgc cggaaagggc tgctgggacc 120

gggggaagag cggcgtctgg aggtgactca gttaagaaga cgaaaccacg ccaccgctgg 180

cggccaggga ctgtagcgct gcgggagatc aggaagtacc agaagtccac tgaaccgctc 240

atcccctttg cgcctttcgt ccgtgtggtg agggagttaa ccaatttcgt aacaaacggg 300

aaagtagagc gctataccgc agaagccctc cttgcgctgc aagaggcagc agaattccac 360

ttgatagaac tgtttgaaat ggcgaatctg tgtgccatcc atgccaagcg tgtcacaatc 420

atgcaaaagg acatacaact tgcaaggcgt atcggaggaa ggcgttgggc a 471

<210> 2

<211> 157

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum-2

<400> 2

Met Ala Ala Thr Leu His Gly Ala Val Ala Leu Thr Ala Gly Leu Pro

1 5 10 15

Leu Leu Leu Leu Gly Pro Gly Ala Ser Gly Gly Ala Ser Thr Ser Ala

20 25 30

Thr Pro Gly Ala Ala Ala Gly Thr Gly Gly Ala Ala Ala Ser Gly Gly

35 40 45

Ala Ser Val Leu Leu Thr Leu Pro Ala His Ala Thr Ala Pro Gly Thr

50 55 60

Val Ala Leu Ala Gly Ile Ala Leu Thr Gly Leu Ser Thr Gly Pro Leu

65 70 75 80

Ile Pro Pro Ala Pro Pro Val Ala Val Val Ala Gly Leu Thr Ala Pro

85 90 95

Val Thr Ala Gly Leu Val Gly Ala Thr Thr Ala Gly Ala Leu Leu Ala

100 105 110

Leu Gly Gly Ala Ala Gly Pro His Leu Ile Gly Leu Pro Gly Met Ala

115 120 125

Ala Leu Cys Ala Ile His Ala Leu Ala Val Thr Ile Met Gly Leu Ala

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Ile Gly Leu Ala Ala Ala Ile Gly Gly Ala Ala Thr Ala

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<210> 3

<211> 411

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atggcccgca cgaagcagac ggcgcgcaag tcgacgggcg gcaaggcgcc ccgcaagcag 60

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cgcttccgcc ccggcaccgt cgcgctccgg gagattcgca agtaccagaa gagcacggag 180

ctgctcatcc gcaagctgcc cttccagcgc ctcgtccgtg agatcgcgca ggatttcaag 240

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Met Ala Ala Thr Leu Gly Thr Ala Ala Leu Ser Thr Gly Gly Leu Ala

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Pro Ala Leu Gly Leu Ala Thr Leu Ala Ala Ala Leu Ser Ala Pro Ala

20 25 30

Thr Gly Gly Val Leu Leu Pro His Ala Pro Ala Pro Gly Thr Val Ala

35 40 45

Leu Ala Gly Ile Ala Leu Thr Gly Leu Ser Thr Gly Leu Leu Ile Ala

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Leu Leu Pro Pro Gly Ala Leu Val Ala Gly Ile Ala Gly Ala Pro Leu

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Ala Gly Ala Thr Leu Val Gly Leu Pro Gly Ala Thr Ala Leu Cys Ala

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gtgaggggagt taaccaattt cgtaacaaac gggaaagtag agcgctatac cgcagaagcc 300

ctccttgcgc tgcaagaggc agcagaattc cacttgatag aactgtttga aatggcgaat 360

ctgtgtgcca tccatgccaa gcgtgtcaca atcatgcaaa aggacataca acttgcaagg 420

cgtatcggag gaaggcgttg ggca 444

<210> 6

<211> 148

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum-6

<400> 6

Met Ala Ala Thr Leu Gly Thr Ala Ala Leu Ser Thr Gly Gly Leu Ala

1 5 10 15

Pro Ala Leu Gly Leu Ala Thr Leu Ala Ala Ala Leu Ser Ala Pro Ala

20 25 30

Thr Gly Gly Val Leu Leu Pro His Ala Pro Ala Pro Gly Thr Thr Ser

35 40 45

His Ala Thr Ala Pro Gly Thr Val Ala Leu Ala Gly Ile Ala Leu Thr

50 55 60

Gly Leu Ser Thr Gly Pro Leu Ile Pro Pro Ala Pro Pro Val Ala Val

65 70 75 80

Val Ala Gly Leu Thr Ala Pro Val Thr Ala Gly Leu Val Gly Ala Thr

85 90 95

Thr Ala Gly Ala Leu Leu Ala Leu Gly Gly Ala Ala Gly Pro His Leu

100 105 110

Ile Gly Leu Pro Gly Met Ala Ala Leu Cys Ala Ile His Ala Leu Ala

115 120 125

Val Thr Ile Met Gly Leu Ala Ile Gly Leu Ala Ala Ala Ile Gly Gly

130 135 140

Ala Ala Thr Ala

145

<210> 7

<211> 711

<212> DNA/RNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum-7

<400> 7

atgttgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60

gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120

cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180

ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240

cccgccgaca tccccgacta cctcaagctg tccttccccg agggcttcaa gtggaggcgc 300

gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360

ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgtc 420

atgcagaaga agaccatggg ctgggaggcc tcctccgagc gcatgtaccc cgaggacggc 480

gccctgaagg gcgagatcaa gcagcgcctg aagctgaagg acggcggcca ctacgacgct 540

gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600

aacatcaagc tcgacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660

cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta a 711

<210> 8

<211> 236

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum-8

<400> 8

Met Leu Ser Leu Gly Gly Gly Ala Ala Met Ala Ile Ile Leu Gly Pro

1 5 10 15

Met Ala Pro Leu Val His Met Gly Gly Ser Val Ala Gly His Gly Pro

20 25 30

Gly Ile Gly Gly Gly Gly Gly Gly Gly Ala Pro Thr Gly Gly Thr Gly Thr

35 40 45

Ala Leu Leu Leu Val Thr Leu Gly Gly Pro Leu Pro Pro Ala Thr Ala

50 55 60

Ile Leu Ser Pro Gly Pro Met Thr Gly Ser Leu Ala Thr Val Leu His

65 70 75 80

Pro Ala Ala Ile Pro Ala Thr Leu Leu Leu Ser Pro Pro Gly Gly Pro

85 90 95

Leu Thr Gly Ala Val Met Ala Pro Gly Ala Gly Gly Val Val Thr Val

100 105 110

Thr Gly Ala Ser Ser Leu Gly Ala Gly Gly Pro Ile Thr Leu Val Leu

115 120 125

Leu Ala Gly Thr Ala Pro Pro Ser Ala Gly Pro Val Met Gly Leu Leu

130 135 140

Thr Met Gly Thr Gly Ala Ser Ser Gly Ala Met Thr Pro Gly Ala Gly

145 150 155 160

Ala Leu Leu Gly Gly Ile Leu Gly Ala Leu Leu Leu Leu Ala Gly Gly

165 170 175

His Thr Ala Ala Gly Val Leu Thr Thr Thr Leu Ala Leu Leu Pro Val

180 185 190

Gly Leu Pro Gly Ala Thr Ala Val Ala Ile Leu Leu Ala Ile Thr Ser

195 200 205

His Ala Gly Ala Thr Thr Ile Val Gly Gly Thr Gly Ala Ala Gly Gly

210 215 220

Ala His Ser Thr Gly Gly Met Ala Gly Leu Thr Leu

225 230 235

<210> 9

<211> 20

<212> DNA/RNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum-9

<400> 9

atggcccgca cgaagcagar 20

<210> 10

<211> 20

<212> DNA/RNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum-10

<400> 10

cctcggggaa ggacagcttc 20

<210> 11

<211> 23

<212> DNA/RNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum-11

<400> 11

acggaaggag taagaggatg ttt 23

<210> 12

<211> 20

<212> DNA/RNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum-12

<400> 12

cggtagtccttcccgttcac 20

<210> 13

<211> 22

<212> DNA/RNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum-13

<400> 13

gcaaagtctg ccgccttaca ac 22

<210> 14

<211> 24

<212> DNA/RNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum-14

<400> 14

tgttatccgc tcacaattcc acac 24

Claims (2)

1. A method for increasing the induction rate of a maize haploid inducer line by genetically engineering a CENH3 protein, the method comprising the steps of:

The method comprises the steps of firstly, replacing the sequence of the N end in a corn CENH3 gene with the N end sequence of a corn H3 gene to obtain a preliminary modified gene 1, wherein the modified gene 1 is shown as SEQ ID NO.5, cloning the gene 1 into a plant overexpression vector pCAMBIA3301-RFP taking a Ubiquitin as a promoter and containing a large molecular weight tag, constructing a vector UBI for over-expressing a centromere specific fusion protein, namely M-tailswap-RFP, and transforming the overexpression vector into a corn receptor to obtain a positive transformant strain LH244 ^{M-tailswap-RFP};

Step two, hybridizing the positive transformant line with a corn receptor parent CAU5 to obtain a hybrid generation 1, then continuously backcrossing 2 generations by taking the CAU5 as a recurrent parent, then selfing for at least 4 generations, and finally forming a novel induction line with high induction rate;

In the first step, the specific fusion protein of the centromere is specifically a fusion expression protein which is formed by replacing the N-end amino acid sequence of the specific histone CENH3 of the maize centromere with the N-end amino acid sequence of the small histone H3 of the maize kernel and connecting a large molecular weight fluorescent tag RFP at the C end of the CENH3 protein; the transformation adopts an agrobacterium embryo leaching method; the corn receptor line adopts LH244 line;

In the second step, the acceptor parent corn is a Stock 6-derived corn haploid induction line CAU5.

2. The method for increasing the induction rate of a maize haploid inducer by genetically engineering a CENH3 protein of claim 1, wherein in step one, the large molecular weight tag is RFP fluorescent protein.