CN106609280B - Application of pollen development related acyl-CoA ligase or coding gene thereof and method for cultivating plant sterile line - Google Patents

Abstract

The invention discloses a method for cultivating a plant sterile line, which comprises the step of reducing the expression or activity of acyl-CoA ligase related to pollen development in plant plants. And a method for transforming a plant from sterile to fertile comprising the step of reducing the rate of synthesis of lipids required by the pollen wall and/or retarding the rate of pollen development. In addition, the invention provides the application of the acyl-CoA ligase related to pollen development or the coding gene thereof in cultivating a plant sterile line or preparing a reagent or a kit for cultivating the plant sterile line. The invention provides a method for creating a plant sterile line by reducing the expression or activity of acyl-CoA ligase associated with pollen development in a plant; a method for transforming the trait of a sterile plant into fertility by delaying the rate of pollen development is provided.

Description

Application of pollen development related acyl-CoA ligase or coding gene thereof and method for cultivating plant sterile line

Technical Field

The invention relates to the technical field of agriculture and biology, in particular to a method for creating a photo-thermo-sensitive sterile line and application thereof in plant breeding.

Background

In agricultural production, as the traditional castration method is time-consuming and labor-consuming, the male sterile line has great advantages in hybrid seed production and agricultural yield improvement. Male sterility is often classified into Cytoplasmic Male Sterility (CMS) and nuclear male sterility (GMS). Establishment of the three-line hybrid system relies on cytoplasmic male sterility. However, there are several disadvantages that prevent the widespread use of three line mating hybridization in practice. Firstly, the cytoplasmic male sterile plants generally have the problem of poor quality; secondly, the combined yield-increasing potential of the three-line hybrid rice is smaller and smaller. Thirdly, as the wild sterile type male sterile cytoplasm is single, once cytoplasmic sterility is lost or some destructive insect pests occur, huge loss can be caused. Along with the discovery of light-temperature sensitive conditional male sterility in nuclear male sterility, the hybrid rice by the two-line method is produced. Compared with the three-line hybridization method, the photo-thermo-sensitive sterile line has two states of a sterile line and a maintainer line. Compared with the three-line method, the two-line method is not limited by the restoring and maintaining relationship, the nuclear sterility can be hybridized with a large amount of conventional varieties, and the pairing is free, so that the hybrid advantage with excellent characters can be obtained more easily, and the problem of the simplification of the male sterile cytoplasm in the three-line method is solved fundamentally. In recent years, the application of two-line hybrid rice in Chinese agricultural production is more and more extensive.

As early as 1973, Shimingsong selectively bred a light-sensitive sterile line from late japonica rice variety (Oryza sativa ssp. japonica) agricultural cultivation 58 in Hubei province of China and provided a new way for heterosis utilization of rice for one line of dual purposes. Subsequently, with agricultural reclamation 58S (NK58S) as the male parent, peruvian 64S (PA64S) obtained by crossing with indica rice has also been widely used in two-line crossing. But fertility of culture 64S is more sensitive to temperature changes. In rice, photo-thermo-sensitive sterile lines are controlled by monogenic recessive loci. Recent researches show that the sterile characters of agricultural reclamation 58S and culture dwarf 64S are controlled by the same genetic locus, and the locus is influenced by temperature and illumination, so that the molecular mechanism of photo-thermo-sensitive fertility conversion is difficult to understand.

So far, thirteen photo-thermo-sensitive sterile lines are found in rice: pms1, pms2, pms3, rpms1, rpms2, tms1, tms2, tms3, tms4, tms5, tms6, rtms1 and Ms-h are located on chromosome 7,3,12,8,9,8,7,6,2,2,5,10 and 9, respectively. Photo-thermo-sensitive sterility has also been reported in tomato, corn and wheat. Recent studies found that a mutant small RNA (small RNA), osa-smR5864m, resulted in the sterile phenotype of pms2 and p/tms2-1 (Nongkail 58S and Pepper 64S) mutants. However, the molecular mechanism of photo-thermo-sensitive infertility is still unclear, so that people lack effective theoretical support and technical means to solve the practical problems in two-line breeding.

In view of incomparable advantages of small genome, rapid growth cycle, a large amount of mutant libraries and the like of arabidopsis, arabidopsis becomes a model plant in the field of plant biological research. In addition, Arabidopsis thaliana can be cultured in a narrow space under strictly controlled conditions such as temperature and light. Previous studies found that some arabidopsis conditional sterile mutants, such as the PEAMT gene mutant t365 and the ms33 mutant with hindered GA/IAA biosynthesis, all showed temperature-sensitive sterile phenotypes.

However, there is still a lack in the art of plant sterile lines with simple control methods for plant breeding, so there is an urgent need for plant sterile line breeding techniques with simple control methods.

Disclosure of Invention

The invention aims to provide a method for cultivating a plant photo-thermo-sensitive sterile line, which comprises the step of creating a photo-thermo-sensitive plant material by reducing the expression or activity of acyl-coenzyme A ligase related to pollen development, thereby solving the problems of few photo-thermo-sensitive genetic loci and low seed production purity of the existing nuclear sterility.

In a first aspect of the invention, there is provided a method of breeding a plant sterile line comprising the steps of: reducing the expression or activity of an acyl-CoA ligase associated with pollen development in the plant.

In another preferred embodiment, said acyl-coa ligase is involved in lipid metabolism during pollen development of said plant.

In another preferred embodiment, the acyl-CoA ligase forms a thioester bond (acyl-CoA) between a fatty acid and coenzyme a (CoA).

In another preferred embodiment, the "reduction" refers to the reduction of the expression activity of acyl-coa ligase during pollen development in the plant, which satisfies the following condition:

the ratio of A1/A0 is less than or equal to 80 percent, preferably less than or equal to 60 percent, more preferably less than or equal to 40 percent, and most preferably 0 to 30 percent;

wherein A1 is acyl-CoA ligase activity associated with pollen development in the plant; a0 is the enzyme activity of the same acyl-CoA ligase in wild-type plants of the same species.

In another preferred embodiment, the acyl-coa ligase is ACOS5 or a homologous protein thereof.

In another preferred embodiment, the wild-type amino acid sequence of ACOS5 is selected from the group consisting of: 1, 2, 3, 4, 5 and 6.

In another preferred embodiment, the acyl-coa ligase is specifically expressed in a cell, tissue or organ selected from the group consisting of: plant inflorescence and anther.

In another preferred embodiment, the cell or tissue comprises: a felt layer.

In another preferred embodiment, the acyl-coa ligase is specifically expressed during anther development.

In another preferred example, the anther development stage includes a pre-anther formation stage (-3 days to 0 days), an anther formation stage, and a post-anther formation stage (1 to 5 days after anther formation).

In another preferred embodiment, the acyl-coa ligase is specifically expressed at stage 6 of anther development.

In another preferred embodiment, the acyl-coa ligase peaks in expression during pollen meiosis.

In another preferred embodiment, the method of reducing pollen development-associated acyl-coa ligase activity in a plant comprises: the expression level of the gene encoding acyl-CoA ligase is decreased, and/or the acyl-CoA ligase activity is decreased.

In another preferred embodiment, said reduction means that the expression level of pollen development-associated acyl-coa ligase E1 in said plant is from 0 to 80%, preferably from 0 to 60%, more preferably from 0 to 40% of wild type, compared to the expression level of wild type acyl-coa ligase E0; and/or the enzymatic activity A1 of an acyl-CoA ligase associated with pollen development in said plant is 0-80%, preferably 0-60%, more preferably 0-40% of wild type compared to the enzymatic activity A0 of an acyl-CoA ligase associated with pollen development in wild type.

In another preferred embodiment, said reducing acyl-coa ligase activity in plants is effected by a means selected from the group consisting of: gene mutation, gene knockout, gene disruption, RNA interference techniques, or combinations thereof.

In another preferred embodiment, the acyl-coa ligase encoding gene is an ACOS5 gene.

In another preferred example, the ACOS5 gene can encode the amino acid sequence shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4SEQ ID No. 5 or SEQ ID No. 6.

In another preferred example, the method comprises the steps of: reducing the expression level of the ACOS5 gene, deleting the ACOS5 gene and/or mutating the ACOS5 gene in the plant to achieve the reduction of the expression or activity of acyl-CoA ligase in the plant.

In another preferred embodiment, the plant comprises crops, forestry plants and flowers; preferably from the gramineae, leguminosae and cruciferae families, more preferably from rice, maize, sorghum, wheat, soybean or arabidopsis thaliana.

In another preferred embodiment, said plant is selected from the group consisting of: cruciferae (Brassicaceae) plants, Arabidopsis (Arabidopsis) plants or Arabidopsis thaliana (A. thaliana).

In a second aspect of the invention, the application of an acyl-CoA ligase related to pollen development or a coding gene thereof is provided, and the application is used for cultivating a plant sterile line or a reagent or a kit for cultivating the plant sterile line.

In another preferred embodiment, the coding gene is the ACOS5 gene.

In another preferred example, the ACOS5 gene can encode the amino acid sequence shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.

In a third aspect of the invention, there is provided a method of converting a plant from sterile to fertile comprising the steps of: reduce the synthesis rate of lipids required by the pollen wall, and/or delay the development rate of pollen.

In another preferred embodiment, the plant is a plant having a reduced level of expression or activity of an acyl-coa ligase associated with pollen development.

In another preferred embodiment, the plant is a plant sterile line grown according to the method of any one of the preceding claims.

In another preferred example, the method comprises: the development of plants and pollen is delayed.

In another preferred example, the method comprises: reducing the metabolic level of the plant, thereby reducing the synthesis speed of the pollen wall.

In another preferred embodiment, the reduction or delay is achieved by: reducing the ambient temperature at which the plant is growing, reducing the illumination time of the plant, or a combination.

In another preferred embodiment, reducing the ambient temperature at which the plant grows comprises controlling the ambient temperature (average temperature) to 17-22 deg.C, more preferably 17-20 deg.C, such as 17 deg.C, 18 deg.C, 19 deg.C or 20 deg.C.

In another preferred embodiment, the time period for reducing the ambient temperature for plant growth comprises the anther formation stage, the pollen maturation stage and the flowering pollination stage, or 2 weeks before and after.

In another preferred example, the growth temperature of the plants is reduced when the plants are bolting or heading, and the normal temperature cultivation is recovered after the low-temperature cultivation for 3-10 days.

In a fourth aspect of the present invention, there is provided a method of plant breeding comprising the steps of maintaining sterility of a plant; transforming the plant from sterile to fertile; and, maintaining the plants fertile and breeding;

in the step of maintaining the plant sterile, comprising maintaining a plant sterile line grown according to the method of the first aspect of the invention;

in the step of converting the plant from sterile to fertile, the method according to the third aspect of the invention comprises converting the plant from sterile to fertile.

In a fifth aspect of the invention, a plant cell is provided, wherein the plant cell develops into a plant having reduced expression or activity of an acyl-coa ligase associated with pollen development.

In another preferred embodiment, the acyl-coa ligase is ACOS 5.

The invention has the advantages that:

(a) a method is provided for creating a plant sterile line by reducing the expression or activity of an acyl-CoA ligase associated with pollen development in a plant.

(b) A method for transforming the trait of a sterile plant into fertility by delaying the rate of pollen development is provided.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is a picture of the ability to restore fertility to a male sterile acos5 mutant at low temperatures;

a Normal fertile phenotype of wild type plants. Under normal conditions (24 ℃), the pods of the acos5-2, acos5-3 mutant did not contain seeds. Plants of the acos5-2 and acos5-3 mutants fully restored fertility under low temperature conditions (17 ℃).

B. Alexander staining of wild type and acos5 mutant anthers. Wild-type anthers were filled with purple viable pollen at both (24 ℃) and (17 ℃). In the acos5-2 and acos5-3 mutants, (24 ℃) only the green residue was present in the anthers, indicating pollen abortion. However, under the condition of (18 ℃), the mutant pollen returns to normal.

FIG. 2 is a diagram of the structure of the gene encoding an acyl-CoA ligase of ACOS5 according to the present invention;

map-based cloning of acos5 mutants fine localization intervals.

Structural information of acos5 gene and mutation site of mutant.

FIG. 3 is a diagram of a half-thin section analysis of the developmental stage of pollen of a restorer mutant of the present invention;

half-thin sections of anthers of wild type, acos5(24 ℃) and acos5(17 ℃). A-D, M-O is wild type medicine chamber, E-H, P-R is acos5 mutant medicine chamber under 24 ℃, I-L, S-U is acos5 mutant medicine chamber under 17 ℃. No difference was observed between wild type and acos5(24 ℃ or 17 ℃) mutant anthers in the stage 6-7 anthers. In phase 8, the microspores were successfully released from the tetrads in the wild type and acos5(24 ℃ or 17 ℃) mutant chambers. In phase 9, some microspores began to degrade in acos5(24 ℃), whereas microspores in acos5(17 ℃) were identical to the wild type. At stage 10, most of the acos5(24 ℃) microspores vacuolate and degrade. However, most of the recovered microspores were normal in acos5(17 ℃). At stage 11 microspores of acos5(24 ℃) were completely degraded in the chamber, but at 17 ℃, with the exception of individually aborted microspores, a large number of normal pollen grains were present in the acos5 chamber. Mature pollen grains appeared in acos5(17 ℃) at stage 12, but only degraded cell residues were clinging to the inner wall of the chamber at acos5(24 ℃). Ep, epidermal layer; en, inner wall of the chamber; ML, intermediate layer; msp, microspore; PG, pollen grains; t, a tapetum layer; tds, tetrad. Bar is 20 um.

FIG. 4 is a scanning electron microscope analysis of pollen from acos5 mutant and recovered plants of the present invention;

scanning electron microscopy of pollen development for wild type and acos5(24 ℃ or 17 ℃) mutant. Wild type anthers contain many normal pollen and the outer wall structure is normal. Whereas the acos5(24 ℃) mutant has no pollen. The restored plant acos5(17 ℃) also had many mature pollen, but the pollen outer wall structure was still partially abnormal.

FIG. 5 is a transmission electron microscopy analysis of the present invention showing wild type, mutants and restored plant pollen development;

wild type (A-C) and acos5(24 ℃ C.) (D-F) and acos5(17 ℃ C.) (G-I) ultrastructures of pollen development. The tetrads of the wild type and acos5(24 ℃ or 17 ℃) mutants developed normally at stage 7, with primary exowall deposition consistent with that of the wild type. In stage 8, microspores released in wild type form the outer exine layer, while microspores in acos5(24 ℃) begin to vacuolate, and in the recovered plants acos5(17 ℃) form a slightly defective outer exine layer. At stage 9 (i.e., vacuolation), acos5(24 ℃) was completely devoid of pollen exine structure, microspores broken up and with cytoplasmic leakage. In contrast, the microspores of acos5(17 ℃) have a relatively intact pollen exine structure and can develop normally. Ba, columnar structure; i, inner wall; msp, microspore; ne, outer wall inner layer; PC, coating pollen; PG, pollen grains; RM, residual; tc, cap structure. Bars is 5 um.

FIG. 6 shows the recombinant proteins of wild-type ACOS5 and mutant mACOS5, which are expressed prokaryotic;

electrophoresis picture of ACOS5 and mACOS5 induced expression in E.coli. Lanes 1-3 are recombinant proteins of wild-type ACOS5, which are induced without IPTG, induced by IPTG and purified, respectively; 4-6 lanes are recombinant proteins of the mutant mACOS5 induced without IPTG, induced with IPTG and purified respectively; lane 7 is a molecular weight marker.

B. And (3) carrying out western blot detection by using the carrier His recombinant protein.

FIG. 7 is a graph showing that ACOS5 protein has acyl-CoA ligase activity;

in an activity BLANK control (BLANK) system, the content of ATP remaining in the system after enzyme activity reaction is 100%, the remaining amount of ATP of wild-type protein ACOS5 is 60%, and the remaining amount of ATP of mutant protein mACOS5 is 90%, which shows that the enzyme activity is obviously reduced.

FIG. 8 is a graph of the induction of ACOS5 without exposure to temperature;

A. semi-quantitative RT-PCR of ACOS5 gene expression in wild type (24 ℃/17 ℃) and ACOS5-2 and ACOS5-3 mutants (24 ℃/17 ℃) inflorescence RNA.

B. Quantitative RT-PCR of inflorescence RNA ACOS5 gene expression of wild type (24 ℃/17 ℃) and ACOS5-2 and ACOS5-3 mutants (24 ℃/17 ℃).

FIG. 9 is a graph showing the comparison of growth rate of pollen at the swelling stage delayed by low temperature;

A. pollen development can be divided into four stages (microspore release stage, monocyte stage, binuclear cell stage and trinuclear cell stage). The left panel shows the size of the buds at different times. The size of microspore development at different stages is above the right panel. DAPI staining of these microspores revealed their developmental stage and is shown below the right panel.

B. Statistical analysis of the sizes of wild-type microspores at different developmental stages shows that the surface area of the microspores at the developmental stage is rapidly increased.

C. The average growth rate of wild microspores at different external temperatures shows that the development process of the microspores can be obviously slowed down under the low-temperature condition.

FIG. 10 is a comparison of the ability to restore fertility to the acos5 mutant by slowing the time of pollen development;

A. statistics of pollen diameters of the arabidopsis wild type and the chlm-4 mutant at different time periods at normal temperature show that the development of the mutant pollen is slower than that of the wild type.

The double mutant of chlm-4 and acos5 can partially restore fertility at normal temperature.

FIG. 11 is a diagram of protein sequence similarity and evolutionary analysis of ACOS5 gene in different species, wherein the alignment of protein sequences shows that the amino acid sequence of ACOS5 orthologous gene is very conserved, mostly more than 60%, and evolutionary analysis shows that orthologous proteins in different species have clear branches and have common origin in dicotyledonous plants and monocotyledonous plants.

FIG. 12 is a phenotype plot of OsACOS5 mutation in rice resulting in male sterility;

A. structural information of OsACOS5 gene of rice and mutant site.

B. There is a full yellow normal anther in the wild type spike.

Anthers of the osacos5 mutant appeared white and lean.

D. Alexander staining showed full pollen grains in the wild type anthers.

E. Alexander staining showed no pollen grains in the mutant anthers.

Detailed Description

The inventor of the present invention has made extensive and intensive studies and, for the first time, has unexpectedly found that, in some specific plant sterile lines, by regulating the expression or activity of acyl-coa ligase related to pollen development, the fertility of the plant can be regulated, and a controllable switch between sterility and fertility is achieved. The inventor also develops various technologies such as corresponding cultivation of plant sterile lines and the like which have wide application values in the aspects of agricultural breeding and the like. The present invention has been completed based on this finding.

In experiments, applicants found that the Arabidopsis ACOS5 gene (Acyl-CoA Synthetase) encodes an Acyl-CoA ligase and is involved in lipid metabolism during pollen development, and that deletion of this gene resulted in mutants that exhibited a completely sterile phenotype under normal growth conditions (24 ℃, 16 hr light/8 hr dark). The low temperature and short sunshine slow down the growth speed and lipid metabolism process of the pollen, so that the mutant overcomes the lipid metabolism defect caused by the deletion of the gene, and the pollen safely passes the rapid expansion period.

The ACOS5 gene encodes an acyl-coa ligase, a conserved protein that exists in different species and has the major function of producing coa esters from long-chain hydroxylated fatty acids (kinenow et al, 2008). In Arabidopsis, previous studies have found that this gene is specifically expressed in the tapetum, and its loss of function results in a male completely sterile phenotype (acos 5-1). Cytological analysis revealed that the mutant pollen wall formed abnormally and lacked sporopollen coverage (deAzevedoSouza et al, 2009). However, no work related to photo-thermo-sensitive infertility by ACOS5 has been reported so far.

The acyl-coa ligase associated with pollen development suitable for use in the present invention is not particularly limited and may be derived from any plant species, representative plants including, but not limited to:

rice (gene number: Os04g 245730 has 64% homology with Arabidopsis thaliana ortholog ACOS5 protein), barley (gene number: MLOC-6813 has 66% homology with Arabidopsis thaliana ortholog ACOS5 protein), alfalfa (gene number: MTR-3 g460770 has 73% homology with Arabidopsis thaliana ortholog ACOS5 protein), tomato (gene number: Solyc02g088710 has 72% homology with Arabidopsis thaliana ortholog ACOS5 protein), grape (gene number: VIT-01 s0010g03720 has 76% homology with Arabidopsis thaliana ortholog ACOS5 protein), and wheat (gene number: TRIUR 3-19356 has 66% homology with Arabidopsis thaliana ortholog ACOS5 protein).

As shown in FIG. 11, the protein sequence similarity and evolutionary analysis of the ACOS5 gene in different species show that the conservation of the gene in different species is strong, and the mutation of the gene in Arabidopsis thaliana can cause sterile traits, so that molecular genetic manipulation of the gene can be used for breeding sterile lines of other species.

The sequence of the Arabidopsis ACOS5 protein is shown as SEQ ID NO. 1, and the nucleotide sequence for coding the Arabidopsis ACOS5 protein is shown as SEQ ID NO. 8. The rice ACOS5 protein sequence is shown in SEQ ID NO. 2, and the nucleotide sequence for coding the rice ACOS5 protein is shown in SEQ ID NO. 9. The barley ACOS5 protein sequence is shown in SEQ ID NO. 3, and the nucleotide sequence for coding the barley ACOS5 protein is shown in SEQ ID NO. 10. The wheat ACOS5 protein sequence is shown in SEQ ID No. 4, and the nucleotide sequence for coding the wheat ACOS5 protein is shown in SEQ ID No. 11. The alfalfa ACOS5 protein sequence is shown in SEQ ID NO. 5, and the alfalfa ACOS5 protein coding nucleotide sequence is shown in SEQ ID NO. 12. The sequence of tomato ACOS5 protein is shown in SEQ ID No. 6, and the nucleotide sequence of tomato ACOS5 protein is shown in SEQ ID No. 13. The grape ACOS5 protein sequence is shown in SEQ ID No. 7, and the nucleotide sequence for coding grape ACOS5 protein is shown in SEQ ID No. 14.

In one aspect, the present invention provides a method of breeding a plant sterile line comprising the steps of: reducing the expression or activity of an acyl-CoA ligase associated with pollen development in the plant.

The terms "acyl-CoA ligase," "acyl-CoA ligase polypeptide," "acyl-CoA ligase protein," and the like, used interchangeably, refer to a protein or polypeptide having an acyl-CoA ligase protein amino acid sequence (e.g., SEQ ID NOS: 1-7). Where not otherwise specified, the term "acyl-coa ligase protein" includes wild-type and mutant acyl-coa ligase proteins.

The acyl-CoA ligase of the present invention may comprise the sequence of amino acids set forth in SEQ ID NOS: 1-7. However, it is not limited thereto because the amino acid sequence of the protein may differ according to the plant species or variety. In other words, it may be a mutant protein or an artificial variant having an amino acid sequence comprising substitution, deletion, insertion or addition of one or several amino acids at one or more positions of the amino acid sequence shown in SEQ ID NO 1-7, as long as it contributes to breeding of a plant sterile line by weakening the activity of the protein. The "several" herein may differ depending on the position or type of the three-dimensional structure of the amino acid residues in the protein, but especially means 2 to 20, especially 2 to 10, more especially 2 to 5. Furthermore, the substitution, deletion, insertion, addition or inversion of amino acids includes those due to artificial variants or natural mutations depending on the individual or kind of plant.

The activity of the acyl-CoA ligase of the present invention can be reduced (attenuated) by: 1) partial or complete deletion of the polynucleotide encoding the protein, 2) modification of expression control sequences to reduce expression of the polynucleotide, 3) modification of sequences on the chromosome or 4) combinations thereof.

In the above, a vector for chromosomal gene insertion can be used to carry out partial or complete deletion of a polynucleotide encoding a protein by replacing a polynucleotide encoding an endogenous target protein with a marker gene or a polynucleotide in which a partial nucleotide sequence is deleted. The length of the "partial" deletion may vary depending on the type of polynucleotide, but is especially from 2bp to 300bp, more especially from 2bp to 100bp, more especially from 1bp to 5 bp.

The expression control sequences may also be modified to reduce polynucleotide expression by: mutations are induced in the expression control sequence by deletion, insertion, conservative or non-conservative substitution of the nucleotide sequence, or a combination thereof to further weaken the activity of the expression control sequence, or to replace the expression control sequence with a less active sequence. Expression control sequences include sequences encoding a promoter, an operator sequence, a ribosome binding site, and sequences which control termination of transcription and translation.

Furthermore, the polynucleotide sequence on the chromosome may be modified to attenuate the activity of the protein by: mutations are induced in the sequence by deletion, insertion, conservative or non-conservative substitution of the nucleotide sequence, or a combination thereof to further attenuate the activity of the sequence, or to replace the polynucleotide sequence with a modified sequence in order to obtain a weaker protein activity.

As used herein, "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide or polypeptide in its native state in a living cell is not isolated or purified, the same polynucleotide or polypeptide is isolated or purified if it is separated from other substances coexisting in its native state.

As used herein, "isolated acyl-coa ligase protein or polypeptide" means that the acyl-coa ligase protein is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify acyl-CoA ligase proteins from plants such as Arabidopsis thaliana rice using standard protein purification techniques. Substantially pure polypeptides are capable of producing a single major band on a non-reducing polyacrylamide gel.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptides of the invention can be naturally purified products, or chemically synthesized products, or using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated or may be non-glycosylated. The polypeptides of the invention may or may not also include an initial methionine residue.

The invention also includes fragments, derivatives and analogs of acyl-coa ligase proteins. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity of a native acyl-coa ligase protein of the invention.

The polypeptide fragment, derivative or analogue of the invention may be:

(i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code;

(ii) polypeptides having a substituent group in one or more amino acid residues;

(iii) a polypeptide formed by fusing a mature polypeptide to another compound (such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol);

(iv) additional amino acid sequences are fused to the polypeptide sequence to form a polypeptide (e.g., a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein).

Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

In a preferred embodiment of the invention, the "acyl-CoA ligase protein" or "acyl-CoA ligase polypeptide" sequence is shown in SEQ ID NO 1-7. The term also includes variants of the sequences of SEQ ID NOS 1-7 that have the same function as an acyl-CoA ligase protein. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of acyl-coa ligase proteins or polypeptides.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to acyl-coa ligase protein DNA under conditions of high or low stringency. The invention also provides other polypeptides, such as fusion proteins comprising an acyl-coa ligase protein or a fragment thereof. In addition to almost full-length polypeptides, the present invention also encompasses soluble fragments of acyl-coa ligase proteins. Typically, the fragment has at least about 10 contiguous amino acids, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the acyl-coa ligase protein sequence.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, "acyl-CoA ligase conservative variant polypeptide" means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are substituted by amino acids having similar or similar properties as compared with the amino acid sequences shown in SEQ ID Nos. 1 to 7 to form a polypeptide. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

TABLE 1

Initial residue(s)	Representative substitutions	Preferred substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region shown in SEQ ID NOS 8-14 or may be a degenerate variant.

As used herein, "degenerate variant" refers in the present invention to nucleic acid sequences which encode a protein having SEQ ID NOs 1-7, but differ from the coding region sequences set forth in SEQ ID NOs 8-14.

Polynucleotides encoding the mature polypeptides of SEQ ID NO 1-7 include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

In a preferred embodiment, the coding sequence for an acyl-coa ligase polypeptide is selected from the group consisting of: (1) a polynucleotide sequence encoding the polypeptide as set forth in SEQ ID NO 1-7; (2) the polynucleotide sequence shown as SEQ ID NO. 8-14; (3) a polynucleotide complementary to the polynucleotide sequence of (1) or (2).

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above polynucleotides which encode polypeptides having the same amino acid sequence as the present invention or fragments, analogs and derivatives of the polypeptides. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" refer to (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. Moreover, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides shown in SEQ ID NOS: 1-7.

The invention also relates to nucleic acid fragments which hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides in length. The nucleic acid fragments can be used in nucleic acid amplification techniques (e.g., PCR) to determine and/or isolate polynucleotides encoding acyl-CoA ligase proteins.

The full-length nucleotide sequence or its fragment of the acyl-CoA ligase protein of the present invention can be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The present invention also relates to vectors comprising the polynucleotides of the present invention, as well as genetically engineered host cells transformed with the vectors or GDSL protein-encoding sequences of the present invention, and methods for producing the polypeptides of the present invention by recombinant techniques. The polynucleotide sequence of the present invention can be used to express or produce recombinant rice acyl-CoA ligase protein by conventional recombinant DNA techniques (Science, 1984; 224: 1431). Generally, the following steps are performed: (1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding an acyl-coa ligase protein, or with a recombinant expression vector comprising the polynucleotide; (2) a host cell cultured in a suitable medium; (3) isolating and purifying the protein from the culture medium or the cells.

In the present invention, the acyl-coa ligase protein polynucleotide sequence may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector well known in the art. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing acyl-coa ligase protein-encoding DNA sequences and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli. Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples are: escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, and the like.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene.

It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells. Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The transformed plant may also be transformed by Agrobacterium transformation or gene gun transformation, such as leaf disk method. Transformed plant cells, tissues or organs can be regenerated into plants by conventional methods to obtain plants with altered tolerance.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

A part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a microarray or a DNA chip (also called a "gene chip") for analyzing the differential expression analysis of genes in tissues. The acyl-CoA ligase protein transcripts can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification using acyl-CoA ligase protein specific primers.

The main advantages of the invention include:

(a) a method is provided for creating a plant sterile line by reducing the expression or activity of an acyl-CoA ligase associated with pollen development in a plant.

(b) A method for transforming the trait of a sterile plant into fertility by delaying the rate of pollen development is provided.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: a laboratory manual (New York: Cold Spring harbor laboratory Press,1989) or a Plant Molecular Biology-laboratory manual (Plant Molecular Biology-laboratory Manual, catalog S.Clark ed, Springer-verlag Berlin Heidelberg,1997), or according to the conditions suggested by the manufacturer. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Materials and methods

Plant material and planting

The Arabidopsis material of the invention is a Lersberg erecta background. Mutagenesis and screening of EMS mutants is referred to Zhang et al 2007. Seeds were pregerminated on 0.1% agarose medium for 72 hours at 4 ℃. The plant material is cultured in vermiculite under the following conditions: incubated at 24 ℃ in the light for 16 h/dark for 8 h (normal conditions) until bolting. Then, the bolting plant was transferred to a light incubator for low temperature culture (17 ℃ -22 ℃). For different photoperiods, the bolting plants were cultured in light for 12 hours/dark for 12 hours at room temperature of 24 ℃ respectively; the treatment was carried out in a light culture for 10 hours/dark culture for 14 hours or in a light culture for 8 hours/dark culture for 16 hours.

Cytological analysis

Plant material was photographed with a Nikon digital camera (D-7000). Alexander staining and DAPI staining are referenced to Alex and er, 1969; ross et al, 1996. For the semi-thin slices, the buds at different development stages are selected for fixation and embedded in the Spurr epoxy resin (for a specific method, refer to Zhang et al, 2007). Sections per 1 μm were made using a Powertome XL (RMCProducts, Tucson, Arizona, USA) microtome and stained with toluidine blue. The photographing of the anther sections was performed using an Olympus DX51 digital camera (Olympus, Japan). Fresh stamen and pollen grain materials are wrapped by 8nm gold particles for scanning electron microscopy experiments and observed by a JSM-840 microscope (JEOL, Japan). For transmission electron microscopy experiments, Arabidopsis thaliana was fixed on ice in a fixative (formulation: 0.1M phosphate buffer, pH7.2, containing 2.5% glutaraldehyde). The bud material is further embedded into Resin ('Hard Plus' Embedding Resin, Unite Kingdom) in turn (refer to Zhang et al, 2007 for a specific method). Ultrathin sections (50-70nm) were observed using a JEM-1230 Transmission Electron microscope (JEOL, Japan).

RNA extraction and quantitative RT-PCR

Total RNA was extracted from the floral tissue of mature Arabidopsis thaliana plants using Trizol reagent (Invitrogen, USA). Using poly-dT (12-18) primer; MMLV reverse transcriptase and corresponding reagents 5. mu.g of RNA was inverted to the first cDNA strand (60 min, 42 ℃). The synthesized cDNA strand was used as a template for PCR. Quantitative RT-PCR was detected by the ABI PRISM 7300 system (Applied Biosystems, USA) using SYBR Green I mastermix (Toyobo, Japan). The program parameters for quantitative RT-PCR were: 5 minutes at 95 ℃,10 seconds at 94 ℃ for 40 cycles of denaturation, and 1 minute for extension at 60 ℃ for return. The primers used in the quantitative RT-PCR are listed in the electronic version appendix. beta-Tubulin was used as a control.

Western Blotting hybridization

Protein samples were subjected to 10% SDS-PAGE and subjected to 110V constant pressure electrophoresis for 90 min. After electrophoresis, the membrane is electrotransferred by a semidry method, the PAGE gel which is good in electrophoresis is cut off from the electrophoresis plate, and is carefully transferred to the nitrocellulose membrane on the membrane transferring plate. The membrane needs to be first saturated with methanol. The membrane transferring plate is sequentially placed in the order of filter paper, membrane, gel and filter paper from bottom to top, and bubbles are removed. The membranes were spun overnight at a constant current of 15 mA. The membrane was transferred to TBST containing 5% skimmed milk powder for 1h blocking. Primary antibody (diluted in TBST at a certain ratio) was added and the hybridization was carried out at 4 ℃ for 1 h. The membrane was washed with TBST 4 times for 15min each. Add secondary antibody (diluted in TBST at appropriate ratio) and hybridize for 1h at room temperature. Washing membranes with TBST for 15 min/4 times. Adding developing solution, and taking the picture with a developing instrument for storage.

Protein expression and purification

2. mu.l of the strain preserved at-70 ℃ was inoculated into 5ml of the corresponding resistant LB medium and cultured overnight at 37 ℃. 100 mul of the strain recovered by overnight culture is taken and inoculated into 100ml of corresponding resistant LB culture medium for shaking culture at 37 ℃ for 4-5h until OD600 is 0.4-0.5. 100ul of 1M IPTG was added and induction was carried out 18 ℃ overnight. The bacteria were collected by centrifugation at 12000g for 2min at 4 ℃. Repeatedly freezing and thawing (-70 deg.C freezing, 37 deg.C thawing) for 5-6 times, disrupting bacteria, 4 deg.C 12000g for 20min, collecting supernatant, filtering with 0.45 μm filter membrane, and storing at-20 deg.C for use. The purification column was prepared and the ethanol in the column was washed with 3-5 bed volumes of ddH 2O. Equilibrating the column, equilibrating the column with at least 5 bed volumes of Binding Buffer, passing the sample through the column at a flow rate of 1ml/min, passing the protein sample to be purified slowly through the column, but not at an excessive rate, and maintaining a drop-by-drop flow through the column. Washing, the column was washed with 10-15 bed volumes of WashBuffer at a flow rate of 1 ml/min. Eluting, eluting the protein with an Elution Buffer of 5 bed volumes, which is preferably less than one bed volume, to concentrate the protein, and collecting the eluted protein for storage at-20 deg.C.

Enzyme Activity assay

The activity of wild-type ACOS55 and mACOS5 proteins was tested by the Luciferase-Based Assay method. The enzyme-catalyzed reaction comprises two steps: acid + ATP- - -acyl-AMP + PPi; Acyl-AMP + CoA- - -Acyl-CoA + AMP; the entire reaction consumes one molecule of ATP. The Luciferase and the Luciferase react to emit fluorescence, the ATP provides energy required by the reaction, and the intensity of the fluorescence emitted by the Luciferase reaction is in direct proportion to the amount of the ATP in the reaction system. The content of the residual ATP in the system after the 4-CL enzyme activity reaction is detected through the fluorescence reaction, so that the enzyme activity of the 4-CL protein to be detected is reflected. The method comprises the following operation steps: 1. melting the protein to be detected on ice, adding a proper amount of protein into Mix1, uniformly mixing, and standing for 30min at room temperature. 2. At various time points, 2. mu.l of the reacted solution from step 1 was added quickly to the prepared Mix2 and mixed well. 3. The fluorescence intensity of the reacted Mix2 was measured with a Lumat LB 9501 luminometer and the measurements were recorded. 4. According to the fluorescence intensity, the Luciferase activity of different samples is prepared as a graph, and the 4-CL activity is inversely proportional to the graph.

The reaction system is as follows:

4-CL catalytic reaction System (200. mu.l) (Mix 1):

luciferase assay reaction (200 μ l) (Mix 2):

example 1 sterile phenotype of Arabidopsis thaliana acos5-2 and acos5-3 mutants was restored under low temperature conditions

The inventors isolated acos5-2 mutants from the arabidopsis Ler ecotype by means of EMS chemical mutagenesis. Meanwhile, a mutant acos5-3 with one exon inserted is screened from a T-DNA insertion mutant library of an arabidopsis Col ecotype. At normal ambient temperatures (24 ℃), the homozygous acos5-2 mutant grew normally but with loss of fertility, with only short seedless pods, as shown in fig. 1A. Genetic analysis shows that the acos5-2 mutant belongs to sporophyte male sterility and is controlled by a monogenic recessive locus. Applicants cultured the acos5-2 mutant at 24 ℃ to bolting and transferred to 18 ℃ for continuous culture, with subsequent pods restored fertility as shown in fig. 1A, whereas the acos5-3 mutant had more difficult restoration of fertility at low temperatures than the point mutation acos 5-2. Wild type plants were not affected under the same low temperature conditions (results not shown). Alexander staining showed that pollen from the acos5-2 and acos5-3 mutants stained purple under cold conditions, as in the wild type, as shown in FIG. 1B, while anthers from the acos5-2 and acos5-3 mutants under normal conditions showed no viable pollen, as in FIG. 1B. This result suggests that cold temperatures can compensate for the defect in male gametophyte development in the acos5-2 and acos5-3 mutants.

Example 2 Gene mapping of ACOS5-2 and ACOS5 Gene Structure

By utilizing the arabidopsis thaliana In/Del molecular marker In the laboratory, the ACOS5 gene is positioned on the first chromosome of arabidopsis thaliana and is closely linked with the molecular marker F7A10 through the primary positioning of sterile phenotype plants In the generation F2 of ACOS5-2 mutant. Six pairs of fine positioning primers are designed afterwards: F24O1, F23N19, F16P17(8), F16P17(Snp-1), F16M19 and F13O11, and further reduces the localization interval of the ACOS5 gene to be within 44KB between two molecular markers of F16P17(23283) and F16P17(23327), wherein the interval comprises 6 candidate genes, as shown in A in FIG. 2. Wherein the At1g62940 gene encodes an acyl-coa ligase protein. In the acos5-2 mutant, a single nucleotide mutation of CCA to CTA (proline mutated to leucine) was detected in the third exon of the gene, as shown in B in fig. 2. For the T-DNA insertion mutant acos5-3, applicants used TAIL-PCR to sequence the amplified border sequence and found that T-DNA insertion was present in the second exon of the acos5-3 mutant, as shown in B in FIG. 2. Applicants further validated using genetic complementation experiments. An At1g62940 genomic fragment was cloned, including an upstream promoter region and a downstream region, using Agrobacterium-mediated transformation into plants heterozygous for acos 5-2/+. 2 of the 5 transgenic lines were identified as acos5-2/acos5-2 background, and these plants restored fertility at normal temperature (24 ℃) to form normal pods (results not shown).

Example 3 Low temperature makes it possible to compensate for the microspore development deficiency in the acos5 mutant

To determine the defect of the acos5 mutant in pollen development, applicants performed anther semi-thin sections. In wild type, microsporocytes undergo meiosis to form tetrads at stages 6 and 7 (Sanders et al, 1999), as shown in fig. 3 a-B. Subsequently, microspores were released from the tetrads and a trinuclear pollen grain with normal pollen walls was gradually formed (FIGS. 3C-D, M-O). No visible differences were observed between the acos5 mutant under ambient conditions (24 ℃) until stage 7 of anther development between the mutant and wild type, indicating that the mutant male gametophyte meiosis was unaffected (FIG. 3F). By the 8 th stage of anther development, acos5 microspores were released from tetrads, and some microspores began to vacuolate (fig. 3G). At stage 10, most of the acos5 microspores began to degrade, as shown by P in fig. 3, and subsequently the cytoplasm of the microspores shrunk and collapsed. Eventually, no normal pollen is formed in the chamber, as shown by R in FIG. 3. On the other hand, in the cold state (17 ℃), the acos5 microspores, after release from tetrads at stage 8, tended to be normal compared to the wild type, and finally produced normal mature pollen grains in the chamber at low temperature, as shown by T-U in FIG. 3, with the exception of a small portion of aborted pollen.

Scanning electron microscopy showed that the acos5-2 mutant had no pollen grains in the chamber at ambient temperature (24 ℃), but the number of pollen grains at low temperature (17 ℃) was substantially the same as that of wild type, with slight abnormalities in the pollen wall structure (FIG. 4). Transmission electron microscopy showed that the structure of the pollen outer wall of the acos5-2 microspore was restored at low temperature. In the tetrad stage, the acos5-2 microspore plasma membrane wave-like fluctuations were normal compared to the wild type under different conditions, as shown in fig. 5 a, D, G. During microspore release, the wild type forms more normal columnar and canopy structures constituting the pollen exine (FIG. 5B), while the microspores of acos5-2 lack normal pollen exine structures at normal temperature (24 ℃), which results in late-stage rupture degradation of the microspores (FIGS. 5E-F). Under the condition of low temperature (17 ℃), the pollen outer wall of the acos5-2 microspore can gradually form a relatively normal rod-shaped and roof structure (shown as H-I in figure 5, which shows that the low temperature can overcome the defect of outer wall synthesis caused by acos5-2 mutation.

Example 4 the mutant protein mACOS5 has reduced acyl-CoA ligase activity and expression is not temperature induced

In order to detect the enzyme activity of the ACOS5 protein, the applicant utilized a prokaryotic expression system to perform expression and purification on the protein. Since the ACOS5-2 mutant is a point mutation, the applicant clones both wild-type ACOS5 and mutant mACOS5 genes for prokaryotic expression (shown in A in FIG. 6 and identified by Western blotting hybridization using His antibody (FIG. 6B). The reaction catalyzed by acyl-CoA ligase requires energy coupling with ATP hydrolysis, so I can indicate the enzyme activity by detecting the amount of ATP remaining in the reaction system. thus the Luciferase activity is inversely proportional to the acyl-CoA ligase activity. the detection of the enzyme activities of ACOS5 and mACOS5 shows that the mutated mACOS5 protein in ACOS5-2 does retain a part of the enzyme activity (shown in FIG. 7).

To elucidate the mechanism of temperature sensitivity of ACOS5 mutants, applicants examined the transcription levels of ACOS5 in wild-type and mutant buds under different temperature conditions. RT-PCR assays showed no significant difference in transcription levels between wild-type and ACOS5-2 mutants of ACOS5 under ambient (24 ℃) and low temperature (17 ℃) conditions, but not in the T-DNA insertion mutant ACOS5-3, as shown in A in FIG. 8. Further quantitative PCR revealed that in the wild type and ACOS5-2 mutant, the ACOS5 gene was expressed slightly higher at low temperature (17 ℃) than at normal temperature (24 ℃) but not at ACOS5-3, as shown in B in FIG. 8. Given that fertility of ACOS5-3 is harder to restore than ACOS5-2, these results indicate that expression and enzyme activity of ACOS5 are critical for pollen wall formation and normal pollen development.

Example 5 lower ambient temperature leads to a reduction in the pollen development rate of Arabidopsis thaliana

The above cytological observations indicate that most of the acos5 mutant microspores are degraded at ambient temperature (24 ℃) during the ring vacuole phase. Early literature suggests that Arabidopsis pollen is growing in volume during maturation. The Applicant speculates that in the acos5 mutant, microspores are not as goodCan bridge the rapid swelling process to cause pollen rupture and abortion. Thus, applicants determined the surface area of wild-type microspores during male gametophyte development, and initially divided the pollen development process into 4 stages depending on the size of the buds: microspore release stage (reieasured stage), monocytic pollen stage (uninduced stage), binuclear pollen stage (bicellular stage) and trinuclear pollen stage (tricellular stage) (FIG. 9, panel A. statistics show that the surface area of microspores after release from tetrads is approximately 500um². After the first mitotic division, the surface area of the microspores in the binuclear stage is enlarged by about 2 times. When the trinuclear pollen is formed, the pollen surface area is increased to 2300um²As shown at B in fig. 9. Meanwhile, the applicant counted the growth rate of each period at different temperatures, and the results showed that the growth rate of microspores from the microspore release period to the monogenic pollen period under the normal temperature condition (24 ℃) was about 8.6um²(ii)/hr; the growth rate of microspore from the mononuclear pollen stage to the binuclear pollen stage is about 38.9um²/hr, cytological analysis showed that most of the disruption of the acos5 microspores was present at this stage; the speed from the binuclear pollen stage to the trinuclear pollen stage is 33.7um²And/hr. However, under the low temperature condition (17 ℃), the growth rate of the second stage decreased by about 3 times as shown by C in fig. 9. The growth rates were determined to be similar in the wild type and acos5 mutants.

Example 6 reduction of pollen development speed partially restores the sterile phenotype of the acos5 mutant

The point mutant of the Arabidopsis magnesium protoporphyrin IX methyltransferase gene chlm-4 shows a phenotype of yellowing of the plant, but the fertility of the mutant is normal (Cao et al, 2010). under the condition of normal temperature (24 ℃), the microspore development process of the chlm-4 mutant is obviously slower than that of the wild type, as shown in A in FIG. 10. With the immediate microspore release of wild type and mutant at 0 hours, the data show that 48 hours later, wild type microspores develop from an average of 12.89 μm to 19.44 μm in diameter, while the chlm-4 mutant microspores develop from an average of 12.73 μm to 18.06 μm. After 96 hours, the wild type microspores increased in diameter to 25.03 μm, while the chlm-4 mutant microspores only increased to 22.35 μm, as shown in FIG. 10A. The double mutant of chlm-4 acos5-2 constructed by the applicant further showed a partial fertility restoration phenotype at room temperature, as shown in FIG. 10B. Therefore, this result suggests that the growth development time delayed by low temperature is an important reason for compensating for the microspore defect of acos 5.

Example 7 mutation of OsACOS5 protein in Rice also causes Male sterile phenotype

Based on sequence alignment information in the GenBank database, ACOS5 has almost orthologous proteins in flowering plants. The applicant selects protein sequences of arabidopsis, rice, barley, wheat, tomato, grape and alfalfa to perform homology comparison, and the result shows that the AMP domain is highly conserved in flowering plants. The unrooted evolutionary tree analyzed based on the neighbor-join method showed that orthologous genes in different species had clear branches in dicotyledonous and monocotyledonous plants, as shown in FIG. 11.

Due to the high homology of the ACOS5 protein in different plants, the applicants obtained the T-DNA insertion mutant, OsACOS5, of rice OsACOS5(LOC _ Os04g 245630) which has a premature stop mutation of AAG to TAG in the first exon, as shown in FIG. 12A. The mutant showed a completely sterile phenotype with anthers displaying a shrunken and whiter state compared to the wild type as shown in B in fig. 12, and alexander staining showed no pollen grains visible in the anthers of the osas 5 mutant compared to the purple filled pollen grains of the wild type as shown in C in fig. 12. According to the experimental results, the OsACOS5 protein of rice has the same biological function as Arabidopsis ACOS5, and the rice sterile line can be cultivated by reducing the expression or activity of the OsACOS5 protein in rice.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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Claims (3)

1. a method for converting a plant from sterile to fertile comprising the steps of: mutating the ACOS5 gene to reduce the expression or activity of acyl-CoA ligase related to pollen development in the plant to cultivate a plant sterile line, and reducing the synthesis speed of lipid required by the pollen wall and/or delaying the pollen development speed to convert the plant from non-fertile to fertile in the flowering period, wherein the plant is rice or Arabidopsis thaliana.

2. A method of plant breeding comprising:

(1) maintaining sterility of the plant; (2) transforming the plant from sterile to fertile; and, (3) a step of maintaining the plants fertile and breeding; in said step of maintaining the plant sterile, comprising breeding a plant sterile line for a mutation in the ACOS5 gene that reduces the expression or activity of an acyl-coa ligase associated with pollen development in said plant; in the step of converting the plant from sterile to fertile, the method comprises reducing the rate of synthesis of lipids required by the pollen wall and/or retarding the rate of pollen development during the flowering phase, thereby converting the plant from non-fertile to fertile, wherein the plant is rice or Arabidopsis thaliana.

3. The method of claim 1 or 2, wherein the wild-type amino acid sequence of ACOS5 is selected from the group consisting of: 1, 2, 3, 4, 5 or 6.

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