CN114854786A - A method for improving the induction rate of maize haploid inducible 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 modifying CENH3 protein through genetic engineering, and particularly relates to the field of genetic engineering. The method comprises the steps of replacing the sequence at the N tail end in the corn CENH3 gene with the sequence at the N tail end of the corn H3 gene to obtain a primary modified gene 1, cloning the gene 1 to a plant overexpression vector pCAMBIA3301-In RFP, constructing a vector UBI of over-expression centromere specific fusion protein M-tailswap-RFP, and transforming the over-expression vector into a corn receptor to obtain a positive transformation strain LH244 ^{M‑tailswap‑RFP} (ii) a The positive strain LH244 ^{M‑tailswap‑RFP} Hybridizing with a receptor parent CAU5 for 1 generation, then continuously backcrossing for 2 generations by taking CAU5 as a recurrent parent, and selfing for at least 4 generations to obtain the compound. The culture method can obviously improve the induction rate of the corn haploid induction line, and can improve the efficiency of field haploid identification and simplify the identification process by utilizing the corn haploid induction line containing the fluorescent marker to carry out haploid induction.

Description

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

technical field

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

Background technique

Maize is a model crop that utilizes heterosis, and more than 97% of the sown area of maize in my country uses hybrids. In the utilization of heterosis, the most critical link is the selection of pure lines. There are two commonly used methods: the first is the pedigree method, that is, through self-crossing or backcrossing through continuous multi-bands, 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, which will be within 1-2 years. Pure lines are available. DH breeding plays an increasingly important role in the commercial corn breeding process, and together with molecular breeding technology and transgenic technology, it has become the core technology of modern corn breeding.

A haploid is an individual with the number of gamete chromosomes in a cell. Haploids have only one set of chromosomes, and both recessive and dominant genes can be manifested in contemporary times, so screening for good traits can be carried out in the early generation, and poor traits can be eliminated in time, which is beneficial to yield, resistance and other favorable alleles. Fast aggregation. After the haploid is doubled, the homozygous individual can be obtained directly, without the phenomenon of gene segregation, and the time to obtain the pure line is shorter than that of the conventional breeding pedigree method. 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 use of DH lines can also rapidly generate mapping populations, chromosomal replacement lines, reverse breeding parents, and apomictic engineering, etc.

At present, in the practical application of maize DH breeding, the acquisition of haploid progeny is mainly through the induction of maternal haploid through the haploid induction line derived from maize Stock6 germplasm. The Stock6 maize haploid inducible line was first reported in 1959, with a maternal haploid induction rate of 2.3%-3.2%. In the following decades, maize breeders around the world continued to improve the Stock6 inducible line by crossing or backcrossing, so that the induction rate of the maize haploid inducible line was significantly improved.

In addition to the stock6 germplasm-based haploid induction line selection method, 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 and is a variant of nucleosomal histone H3, which plays an important role in the positioning of the centromere on the chromosome. The current research shows that the use of genetic engineering means to knock out or mutate the CENH3 gene of maize, rice, Arabidopsis and other plants can make the gametophytes or sporophytes of the plants have the ability to induce haploid progeny, thereby Create haploid inducible lines.

However, there are still obvious defects in the breeding or creation methods of the above two haploid induction lines. For the method of breeding maize haploid inducible lines by improving Stock6 germplasm, there are mainly the following shortcomings: 1. Even after years of backcross breeding, the induction rate of haploid inducible lines is still slow to increase. 2. Due to the limitation of germplasm resources, the improvement of agronomic traits of haploid inductive lines and the improvement of haploid induction rate cannot be fully balanced, resulting in materials with high induction rate, which may have poor agronomic traits , and cannot be used for commercial breeding applications.

For the method of creating maize haploid inducible lines by genetic engineering of CENH3 gene, there are currently the following disadvantages: 1. The induction rate of haploid inducible lines is still low, and in the created inducible line population, the differences between individual plants The haploid induction rate is unstable. 2. Since the genetic background of most conventional maize lines does not carry the color marker gene, it is difficult to identify the haploids induced by the induced lines created with them.

SUMMARY OF THE INVENTION

Therefore, the present invention provides a method for improving the induction rate of maize haploid inducible lines by genetically engineering CENH3 protein, so as to solve the problems of slow increase in the induction rate of maize haploids, low efficiency of haploid identification, and agronomic traits of inducible lines in existing maize Poor and other issues.

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

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

Step 1, replace the N-terminal sequence of the maize CENH3 gene with the N-terminal sequence of the maize H3 gene to obtain a preliminary modification of gene 1, and then clone the gene 1 into a plant with Ubiquitin (UBI) as a promoter and containing a large molecular weight tag. In the overexpression vector pCAMBIA3301-RFP, a vector UBI:M-tailswap-RFP overexpressing the centromere-specific fusion protein was constructed, and the overexpression vector was transformed into the maize receptor to obtain the positive transformation line LH244 ^{M-tailswap- RFP} ;

In step 2, the positive line is crossed with the recipient parent maize CAU5 for 1 generation, then backcrossed with CAU5 as the recurrent parent for 2 generations, and then selfed for at least 4 generations, and finally a new type of haploid with high induction rate is formed. inductive line.

Further, in the first step, the large molecular weight tag is RFP fluorescent protein.

Further, in the step 1, the centromere-specific fusion protein is specifically by replacing the N-terminal amino acid sequence of the maize centromere-specific histone CENH3 with the N-terminal amino acid sequence of the maize nucleosome histone H3, and in the CENH3 protein. The C-terminus is linked to the fusion expression protein of high molecular weight fluorescent tag RFP.

Further, in the first step, the transformation adopts the Agrobacterium immersion method.

Further, in the first step, the maize acceptor strain is LH244 strain.

Further, in the second step, the recipient parent maize is the maize haploid inducible line CAU5 derived from Stock6.

The cultivation method of the present invention selects the C-terminus of maize CENH3 to splicing the N-terminus of the maize H3 gene and connects the fluorescent protein: the chimeric gene formed by the splicing of H3 and CENH3 is still specifically expressed in the centromere, and it is introduced into maize, The fluorescent label can be stably expressed in the nucleus. In addition, because the CENH3 chimeric protein spliced with the N-terminal sequence of the H3 protein may not have the same function as the wild-type CENH3 protein, and its C-terminal is fused with an exogenous macromolecular fluorescent protein tag RFP, which leads to its integration into the centromere. When it competes with the natural CENH3 protein derived from the mother, it may be due to the lag in the time point of integration into the centromere, or the efficiency of competing spindle filaments is not as good as that of the normal mother. This is a natural CENH3 protein, so chromosomes containing a spliced CENH3 fusion protein (M-tailswap-RFP) are more likely to be lost in mitosis lag.

The present invention has the following advantages:

The cultivation method of the invention significantly improves the induction rate of the maize haploid inducible line, and the maize haploid inducible line with fluorescent markers can not only be used as a haploid inducible line to produce haploid in the offspring, but also can stably express fluorescence Fusion protein, which greatly simplifies the identification process; improves the efficiency of haploid identification in the field and simplifies the identification process.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, other implementation drawings can also be obtained according to the extension of the drawings provided without creative efforts.

The structures, proportions, sizes, etc. shown in this specification are only used to cooperate with the contents disclosed in the specification, so as to be understood and read by those who are familiar with the technology, and are not used to limit the conditions for the implementation of the present invention, so there is no technical The substantive meaning above, any modification of the structure, the change of the proportional relationship or the adjustment of the size should still fall within the technical content disclosed in the present invention without affecting the effect and the purpose that the present invention can produce. within the range that can be covered.

Fig. 1 is a kind of carrier (UBI:M-tailswap) provided by the present invention that replaces the N-terminus of maize CENH3 protein with the N-terminus of maize H3 protein, and connects the red fluorescent protein tag (RFP) at the C-terminus of the chimeric CENH3 protein - schematic diagram of the construction of RFP);

Fig. 2 is the identification result of the transgenic lines using the sequence amplification primers after splicing the N-terminal of H3 and the C-terminal of CENH3 provided by the present invention; wherein, lane 1 and lane 2 are two positive plant DNAs of LH244 ^{M-tailswap-RFP} The biological repeat of , lane 3 is the marker, and the brightest band points to 750bp;

Fig. 3 utilizes real-time fluorescence quantitative PCR (qRT-PCR) provided by the present invention to carry out CENH3 gene expression amount comparison between LH244 ^{M-tailswap-RFP} positive overexpression line and control line;

Fig. 4 is the flow chart of assembling group provided by the invention;

5 is a data comparison diagram of the haploid induction rate of each generation in the process of introducing the UBI:M-tailswap-RFP overexpression vector into the maize CAU5 inducible line through backcross breeding provided by the present invention;

Figure 6 is a comparison diagram of the pollen viability staining of CAU5 provided by the present invention and the newly bred new inductive line CAU5 ^{M-tailswap-RFP} ;

Fig. 7 is the ratio comparison chart of the pollen of 5 kinds of vigor grades of CAU5 provided by the present invention and the newly bred novel induction line CAU5 ^{M-tailswap-RFP} ;

Fig. 8 is a comparison diagram of plant photos of the novel fluorescent induction line CAU5 ^{M-tailswap-RFP} and CAU5 bred provided by the present invention.

Detailed ways

The embodiments of the present invention are described below by specific specific embodiments. Those who are familiar with the technology 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 present invention. , not all examples. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example Method for improving the induction rate of maize haploid inducible lines by transforming the maize CENH3 gene

illustrate:

For the nucleotide sequence of maize CENH3 gene, see Sequence Listing <210>1, NO.1 seq, 2 Ambystoma lateralex Ambystoma jeffersonianum-1; for the amino acid sequence deduced from the nucleotide sequence of maize CENH3 gene, see Sequence Listing <210>2, NO.2 seq, 2 Ambystoma laterale x Ambystoma jeffersonianum-2.

For the nucleotide sequence of maize HISTONE3.2(H3) gene, see Sequence Listing <210>3, NO.3 seq, 2 Ambystomalaterale x Ambystoma jeffersonianum-3; for the nucleotide sequence of maize CENH3 spliced maize H3 gene, see Sequence Listing <210> 4, NO.4 seq, 2 Ambystoma laterale x Ambystoma jeffersonianum-4.

For the nucleotide sequence of maize CENH3 spliced maize H3 gene, see Sequence Listing <210>5, NO.5 seq, 2 Ambystomalaterale x Ambystoma jeffersonianum-5; for the deduced amino acid sequence of maize CENH3 spliced maize H3 gene, see Sequence Listing <210>6 , NO.6 seq, 2 Ambystoma laterale x Ambystoma jeffersonianum-6.

For the nucleotide sequence of the reporter gene RFP, see Sequence Listing <210>7, NO.7 seq, 2 Ambystoma lateralex Ambystoma jeffersonianum-7; for the deduced amino acid sequence of the reporter gene RFP, see Sequence Listing <210>8, NO.8seq, 2 Ambystoma laterale x Ambystoma jeffersonianum-8.

Sequence amplification primers after splicing of the N-terminus of H3 and the C-terminus of CENH3:

F: 5'ATGGCCCCGCACGAAGCAGA3' (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 sequences

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

R: 5'CGGTAGTCCTTCCCGTTCAC3' (see Sequence Listing <210>12, NO.12seq, 2 Ambystomalaterale x Ambystoma jeffersonianum-12)

Primers for Bar Gene

F: 5'GCAAAGTTCTGCCGCCTTACAAC3' (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)

(1) The vector construction diagram and construction method of the N-terminus of the maize H3 gene splicing the C-terminus of the CENH3 gene and then red fluorescent protein (RFP)

Figure 1 shows the schematic diagram of the construction of the vector that the N-terminus of maize H3 is spliced with the C-terminus of maize CENH3, and then connected to red fluorescent protein (RFP). The specific operation steps are as follows:

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

2. Design a specific primer according to the N-terminal sequence of the corn H3 gene, add the restriction enzyme site XbaI before the forward primer, add the restriction enzyme site SpeI after the reverse primer, and utilize the specific primer to amplify the above-mentioned cDNA by the PCR method. Increase, obtain the target fragment of the splicing H3 gene containing the restriction enzyme site; design specific primers according to the C-terminal sequence of the maize CENH3 gene, add the restriction enzyme restriction site SpeI before the forward primer, and add the restriction enzyme restriction site KpnI after the reverse primer. , and the above-mentioned cDNA is amplified by the specific primer by the PCR method to obtain the target fragment of the splicing CENH3 gene containing the restriction enzyme cleavage site;

3. Utilize restriction endonucleases XbaI/SpeI and SpeI/KpnI to carry out double digestion on the above-mentioned H3 and CENH3 gene fragments respectively, and simultaneously use corresponding restriction endonucleases XbaI/KpnI to carry out double digestion on the pCAMBIA3301-RFP vector, And respectively carry out agarose gel electrophoresis on the enzyme digestion products of each gene fragment and vector and cut the gel for recovery;

4. The gene fragments of H3 and CENH3 and the digested recovery products of the pCAMBIA3301-RFP vector were ligated by T4 ligase, and then the ligated products were transformed into Escherichia coli DH5α strains, and the positive monoclones containing the ligated products were screened by PCR technology. ;

5. Amplify the positive monoclonal colonies, extract the plasmids, and send them to the biological company for sequencing of the target fragments. By comparing the returned sequencing results, the bacterial liquid containing the required correct target gene fragments is carried out. Plasmid extraction for the next step of genetic transformation.

(2) Obtainment of transgenic lines

The genetic transformation of maize adopts the Agrobacterium dipping method, and the maize recipient strain adopts the LH244 strain. The specific operation process is as follows:

1. Transfer the vector plasmid constructed in (1) into the Agrobacterium EHA105 strain, screen the positive monoclonal colonies containing the target vector UBI:M-tailswap-RFP by PCR technology, and propagate the positive Agrobacterium colonies. .

2. Select the ear of the corn LH244 line 10-12 days after self-pollination as the material for callus induction, separate the young embryos from the grains on the corn ear in the ultra-clean bench, and temporarily store the collected young embryos in high Infiltration medium for use;

3. The Agrobacterium bacterial liquid containing the purpose vector UBI:M-tailswap-RFP is cultivated to an OD value equal to 0.8, the thalline is collected by centrifugation, and the thalline is resuspended with a resuspended liquid containing acetosyringone, and the young embryos collected above are collected. Soak in Agrobacterium resuspension for 5 min, then transfer to co-cultivation medium, and cultivate in dark at 25°C for 7 days;

4. Transfer the immature embryos that have gone through the dark culture stage to the gradient screening medium containing resistance, and transfer them once every two weeks, for a total of 3 rounds of screening;

5. Transfer the immature embryos that have been screened and formed callus into differentiation medium, and cultured for 2 weeks to obtain regenerated plants;

6. The regenerated plants are hardened and then transferred to the field or greenhouse for cultivation;

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

8. Extract the total RNA of the leaves of the positively transformed plants, reverse-transcribe them into cDNA, and use qRT-PCR technology to screen the plants with high CENH3 chimeric gene expression and good plant growth status by using qRT-PCR technology combined with the growth status of the field plants. This portion of the selected plants was selfed to obtain homozygous transgenic progeny.

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

After three generations of stable self-crossing of the transgenic lines identified as positive, the pollen of the inductive line CAU5 was used to cross-pollinate the transgenic positive plants and recipient plants, respectively, and the populations of the progeny F1, BC1F1, BC2F1, BC2F2, BC2F3 and BC2F4 generations were counted respectively. Variation trend of induction rate and field agronomic traits.

In the breeding process of each generation, by means of molecular marker-assisted selection, two pairs of primers (the N-terminus of H3 and the C-terminus of CENH3 spliced sequence amplification primer and ZmPLA1-primer sequence) were used to identify the progeny lines. The overexpression vector (UBI:M-tailswap-RFP) of the maize haploid inducible gene locus ZmPLA1 and maize H3 splicing CENH3 was screened and retained, and the identification results are shown in Figure 2; at the same time, in the backcross generation, each generation The lines with the highest haploid induction rate were selected for backcrossing, 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 positive plants identified by PCR

1. Using the obtained transgenic lines, the positive plants identified by the primers of the Bar gene, the target sequences in the positive plants were further determined by using the N-terminal of H3 and the C-terminal of CENH3 after splicing sequence amplification primers.

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

The leaves of the transgenic line LH244 ^{M-tailswap-RFP} and the control group LH244 were taken in the field, and the total RNA was extracted and reverse transcribed to obtain cDNA after storage in liquid nitrogen. Primers were designed with 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). The reaction system is shown in Table 1:

Table 1 Reaction system

component 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 capacity 30

The fluorescence quantitative PCR instrument was ABI7500 from American Applied Biosystems.

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

Calculation method of relative expression: The relative quantification of gene expression was carried out by applying the 2 ^-ΔΔCT method.

The expression level of Actin gene in each sample was set as 1.0 and calculated by subtracting the relative expression level of the gene in the sample. Three technical replicates were performed for each sample and the average was obtained.

3. Identification results

The results are shown in Figures 2 and 3. The positive transformed 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. The expression of CENH3 gene in the positive transformed strain LH244 ^{M-tailswap-RFP} was significantly higher than that of the control strain LH244, as shown in Figure 3.

Experimental example 2 Calculation method and comparison of haploid induction rate

1. Identification method of haploid:

The method used in this experiment to identify haploids is color-coded selection, that is, to identify haploids and diploids by identifying the R-nj markers on the grains. We used this method to determine the induction rates of novel inducers and controls. The specific test method is as follows: when the new inductive line CAU5 ^{M-tailswap-RFP} and the control group CAU5 ^{LH244-Intergressed} are used as male parents respectively, when they are crossed with the conventional line hybrid Zhengdan 958, purple endosperm and purple embryos will be produced on the hybrid ear. (diploid) and purple endosperm non-purple embryos (quasi-haploid). Since the color marking may be difficult to identify on some grains, the identified pseudo-haploids will be planted in the next season, and the actual number of haploids will be screened out through the field performance of the haploids.

Calculation method of haploid induction rate:

The induction rate of an induced line = the number of haploid grains detected on the hybrid ear when the induced line is the male parent/the total number of grains on the hybrid ear.

2. The comparison of induction rate between the new induced line and the control group is shown in Figure 5. 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 induced line CAU5 ^{M-tailswap-RFP} was significantly higher than that of the control induced line CAU5 ^{LH244-introgressed} from the BC2F1 generation; and after entering the selfing generation (BC2F2), the new induced line CAU5 ^M- The haploid induction rate of ^tailswap-RFP increased faster, and the population induction rate in the BC4F4 breeding generation reached about 16.3%, which was significantly higher than that of the control group by about 6.1%.

Experimental Example 3 Calculation Method and Comparison of Pollen Vitality

Pollen Vitality Calculation Method and Judgment Criteria

Prepare a TTC solution with a concentration of 1%, take a mature pollen, gently squeeze out the pollen content on a glass slide, drop a drop of TTC solution in the air, slowly cover it with a cover glass, and let it stand in a 37°C incubator for 5 Minutes later, it was observed under an ordinary phase contrast microscope. The dyeing degree of pollen is divided into 5 grades: the first grade is high vigor, which means that a large area of pollen is dyed red; the second grade is medium vigor, which shows that a large area of pollen is pink and the pollen dyeing intensity is medium; The third grade of pollen is low vigor, which is a large area of pink-white pollen in the pollen; the fourth grade is inactive pollen, which is all pure white pollen; and the fifth grade is aborted grains, as shown in the figure 7 is shown. The proportions of the five types of pollen in the inducible line CAU5 and the new inducible line CAU5 ^{M -tailswap-RFP} were counted respectively. At least 10 pollen from different locations were tested for each line. Recipe of TTC solution: Weigh 1.0g of chlorotriphenyltetrazolium and dissolve it in 1000mL of pure water, invert it upside down and mix it, then dispense it into a 1mL centrifuge tube, wrap it in tin foil and store it away from light, and pay attention to use Avoid light.

The comparison of the pollen viability staining between CAU5 and the newly bred new inducible line CAU5 ^{M-tailswap-RFP is} shown in Figure 6; The ratio comparison is shown in Figure 7. The results showed that the overall pollen viability of the novel inducible line CAU5 ^{M-tailswap-RFP} was significantly lower than that of its donor parent CAU5, especially the proportion of low viability pollen in the pollen of CAU5 ^{M-tailswap-RFP} was significantly increased, indicating that the induction The pollen viability of the line may have a certain correlation with the haploid induction ability.

Comparison of plant photos between the new fluorescent inducible line selected in experimental example 4 and CAU5

The phenotypic diagram of the plant photos of the selected new fluorescent inducible line and CAU5 is shown in Figure 8. As can be seen from the figure, the new haploid inducible line CAU5 ^{M-tailswap-RFP} cultivated by the method of the present invention, in addition to the haploid induction rate is significantly improved, its agronomic traits are also significantly improved compared with its donor parent CAU5, with a higher The plant height and more developed tassel.

To sum up, the method of the present invention has high operational feasibility, mature technical solutions, and can significantly improve the induction rate of maize haploid inductive lines within a short breeding cycle, and the inventive method will effectively improve the current maize haploid breeding. The efficiency of technology in production practice has important guiding significance.

Although the present invention has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

sequence listing

<110> Shenyang Agricultural University

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

<141> 2022-02-17

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atcaggaagt accagaagtc cactgaaccg ctcatcccct ttgcgccttt cgtccgtgtg 240

gtgagggagt 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 gtgggagcgc 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 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

cctcgggggaa 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

cggtagtcct tcccgttcac 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 (6)

1. A method for improving the induction rate of a corn haploid inducer line by genetically engineering a CENH3 protein, which is characterized by comprising the following steps:

step one, replacing a sequence at the N tail end in a corn CENH3 gene with a sequence at the N tail end of a corn H3 gene to obtain a primary modified gene 1, cloning the gene 1 into a plant over-expression vector pCAMBIA3301-RFP which takes Ubiquitin as a promoter and contains a large molecular weight label to construct a vector UBI, M-tailswap-RFP, of over-expressed centromere specific fusion protein, and transforming the over-expression vector into a corn receptor to obtain a positive transformation strain LH244 ^{M-tailswap-RFP} ；

And step two, hybridizing the positive transformation strain with a corn receptor parent CAU5 to obtain a hybrid 1 generation, then continuing backcrossing for 2 generations by taking CAU5 as a recurrent parent, and then selfing for at least 4 generations to finally form a novel induction line with high induction rate.

2. The method for improving the induction rate of the corn haploid inducer line by genetically engineering the CENH3 protein as claimed in claim 1, wherein in the first step, the large molecular weight tag is RFP fluorescent protein.

3. The method for improving the induction rate of the corn haploid inducer line by genetically engineering the CENH3 protein as claimed in claim 1, wherein in the first step, the centromere-specific fusion protein is a fusion expression protein obtained by replacing the N-terminal amino acid sequence of the corn centromere-specific histone CENH3 with the N-terminal amino acid sequence of the corn nucleosome histone H3 and connecting a high molecular weight fluorescent tag RFP to the C-terminal of the CENH3 protein.

4. The method for improving the induction rate of the corn haploid inducer line by genetically engineering the CENH3 protein as claimed in claim 1, wherein in the first step, the transformation is performed by an Agrobacterium embryo dipping method.

5. The method for improving the induction rate of the corn haploid inducer line by genetically engineering the CENH3 protein as claimed in claim 1, wherein in the first step, the corn acceptor line is the LH244 line.

6. The method for improving the induction rate of the corn haploid induction line by genetically engineering the CENH3 protein as claimed in claim 1, wherein in the second step, the receptor parent corn is the corn haploid induction line CAU5 from Stock 6.

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