CN106883291B

CN106883291B - Plant type related protein PROG2 and encoding gene and application thereof

Info

Publication number: CN106883291B
Application number: CN201510941421.9A
Authority: CN
Inventors: 谭禄宾; 吴永振; 李显然; 刘凤霞; 朱作峰; 付永彩; 顾凭; 孙传清
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2015-12-16
Filing date: 2015-12-16
Publication date: 2019-12-13
Anticipated expiration: 2035-12-16
Also published as: CN106883291A

Abstract

The invention discloses a plant strain related protein PROG2, and a coding gene and application thereof. The plant strain related protein PROG2 provided by the invention is a1) or a2) or a3) as follows: a1) the amino acid sequence is protein shown as a sequence 2 in a sequence table; a2) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 2 in the sequence table; a3) the protein related to the plant type is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in a1) or a 2). Experiments prove that the plant strain related protein PROG2 has very important function for regulating and controlling the plant type and the yield of rice, and has wide prospect in breeding new high-yield rice varieties.

Description

Plant type related protein PROG2 and encoding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a plant strain related protein PROG2, and a coding gene and application thereof.

Background

Rice, one of the most important food crops in the world, breeds over 50% of the world's population. However, recent studies have shown that the narrow genetic background of rice cultivars and the laggard breeding techniques are the main causes of this phenomenon before 78%, 37% and 81% of rice production areas in china, india and indonesia, respectively, have been in the lag from 1961 to 2008 in three producing countries of rice.

The common wild rice is an ancestor of cultivated rice in Asia, and the wild rice is naturally selected and artificially selected in the process of evolving into cultivated rice, so that the gene diversity is reduced and the allele number is reduced. According to statistics, the allelic gene factor of the cultivated rice is about 60 percent of that of wild rice, so that the genetic bottleneck problem of the current rice variety breeding is caused. Therefore, the method is an effective way for solving the current rice breeding problem by discovering and utilizing the excellent domesticated genes which are lost or weakened in the cultivated rice from the genome of the common wild rice Oryza rufipogon Griff of the rice closely related wild species and applying the excellent domesticated genes to the rice breeding production.

The rice plant type is one of the key factors influencing the yield, and the establishment of an ideal plant type is also an important way for rice breeders to improve the rice yield. The wild rice resources in China are rich, and the digging, positioning and cloning of the rice plant type related genes from the wild rice not only contributes to widening the genetic diversity of rice cultivars, but also provides important reference and theoretical basis for modern molecular breeding of rice, and has important significance for strengthening the protection of the wild rice gene resources in China and changing the resource advantages into economic advantages.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the yield of rice.

In order to solve the above problems, the present invention provides a plant strain-related protein.

The plant strain related protein provided by the invention is named as PROG2, is derived from wild rice in east and west of Jiangxi, and is a1) or a2) or a3) as follows:

a1) The amino acid sequence is protein shown as a sequence 2 in a sequence table;

a2) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 2 in the sequence table;

a3) The protein related to the plant type is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in a1) or a 2).

Wherein, the sequence 2 in the sequence table is composed of 188 amino acid residues.

in order to facilitate the purification of the protein in a1), the amino terminal or the carboxyl terminal of the protein shown in the sequence 2 in the sequence table can be connected with a label shown in the table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein PROG2 of a3) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein PROG2 in a3) above may be synthesized artificially, or may be obtained by synthesizing the coding gene and then performing biological expression.

the gene encoding the protein PROG2 in a3) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 1 in the sequence table, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 above at the 5 'end and/or 3' end thereof.

Nucleic acid molecules encoding the PROG2 are also within the scope of the invention.

The nucleic acid molecule encoding the PROG2 can be a DNA molecule represented by (b1) or (b2) or (b3) or (b4) as follows:

(b1) The nucleotide sequence is a DNA molecule shown as a sequence 1 in a sequence table;

(b2) The coding sequence is DNA molecule of 125 th-691 th nucleotide from 5' end of sequence 1 in sequence table;

(b3) A DNA molecule having 75% or more identity to the nucleotide sequence defined in (b1) or (b2) and encoding the protein of claim 1;

(b4) a DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in (b1) or (b2) and which encodes the protein of claim 1.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

wherein, the sequence 1 in the sequence table consists of 892 nucleotides, and the nucleotide of the sequence 1 in the sequence table encodes an amino acid sequence shown as a sequence 2 in the sequence table.

The nucleotide sequence encoding PROG2 of the present invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which have been artificially modified to have 75% or more identity to the nucleotide sequence of PROG2 isolated in accordance with the present invention are derived from and identical to the nucleotide sequence of the present invention as long as they encode PROG2 and are related to plant type.

the term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein consisting of the amino acid sequence shown in sequence 2 of the sequence listing of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

Expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines containing the nucleic acid molecules encoding the PROG2 are also within the scope of the invention.

The expression cassette includes a promoter, a nucleic acid molecule encoding the PROG2, and a terminator. The promoter can be PROG2 promoter (shown in the 1 st to 1258 th positions from the 5' end of the sequence 3); the nucleic acid molecule encoding the PROG2 can be as shown in sequence 3 from 1383 to 1949 from the 5' end; the terminator may specifically be the PROG2 terminator (shown in sequence 3 from positions 2151 to 4214 from the 5' end).

the recombinant vector can be a recombinant plasmid obtained by inserting the encoding gene of PROG2 (namely the DNA molecule shown in the 125 th-691 th site from the 5' end of the sequence 1 in the sequence table) into a starting plasmid through an expression cassette containing the encoding gene of PROG 2.

The recombinant vector can be specifically a recombinant plasmid D81 obtained by replacing a fragment between EcoR I and Sma I recognition sequences of pCAMBIA3301 (pCAMBIA3301 is cut into a large fragment and a small fragment by restriction endonucleases EcoR I and Sma I, and the DNA is the small fragment) with a DNA molecule shown in a sequence 3 in a sequence table, and the recombinant plasmid D81 expresses PROG2 shown in a sequence 2 in the sequence table. The difference between pCAMBIA3301 and D81 is only that the DNA fragment between EcoR I and Sma I recognition sequences of pCAMBIA3301 (pCAMBIA3301 is cut into a large fragment and a small fragment by restriction endonucleases EcoR I and Sma I, and the DNA is the small fragment) is replaced with the DNA molecule shown in sequence 3 of the sequence listing.

The recombinant microorganism can be obtained by introducing the recombinant vector into the starting microorganism.

The starting microorganism may be a yeast, bacterium, algae or fungus. The bacteria may be gram positive or gram negative bacteria. The gram-negative bacterium may be Agrobacterium tumefaciens (Agrobacterium tumefaciens). The Agrobacterium tumefaciens (Agrobacterium tumefaciens) may be Agrobacterium tumefaciens EHA 105.

none of the transgenic plant cell lines includes propagation material. The transgenic plant is understood to include not only the first generation transgenic plant obtained by transforming the recipient plant with the PROG2 gene, but also its progeny. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

The application of the PROG2, the nucleic acid molecule encoding the PROG2, the expression cassette, the recombinant vector, the recombinant microorganism or the transgenic cell line containing the nucleic acid molecule encoding the PROG2 in plant type and/or yield regulation also belongs to the protection scope of the invention.

The application of the PROG2, the nucleic acid molecule encoding the PROG2, the expression cassette, the recombinant vector, the recombinant microorganism or the transgenic cell line containing the nucleic acid molecule encoding the PROG2 in breeding transgenic plants with changed plant types and/or changed yield also belongs to the protection scope of the invention.

In the above application, the yield change may be a decrease in yield of the individual plant.

In the above application, the plant type change may be plant height reduction and/or tillering number increase and/or tillering angle increase and/or primary branch number reduction and/or secondary branch number reduction and/or stem ear number reduction.

In the above application, the plant may be any one of d1) -d 4): d1) a monocot plant; d2) a dicotyledonous plant; d3) rice; d4) rice variety ZH 17.

In order to solve the technical problems, the invention also provides a method for cultivating the transgenic plant.

The method for cultivating the transgenic plant comprises the steps of introducing a nucleic acid molecule for encoding PROG2 into a receptor plant to obtain a transgenic plant; compared with the acceptor plant, the transgenic plant has the advantages of reduced single plant yield and/or reduced plant height and/or increased tiller number and/or increased tiller angle and/or reduced primary stalk number and/or reduced secondary stalk number and/or reduced main stem ear number.

In the above method, the recipient plant may be any one of d1) -d 4): d1) a monocot plant; d2) a dicotyledonous plant; d3) rice; d4) rice variety ZH 17.

Experiments prove that the plant type related protein PROG2 and the coding gene thereof provided by the invention can regulate and control the plant type: t is₀The transgenic PROG2 positive plants show SIL 40-like phenotype; t in comparison with the recipient plant ZH17₀The plant height of the transgenic PROG2 gene positive plant is reduced, the tillering number is increased, the tillering angle is increased, the number of primary branches is reduced, the number of secondary branches is reduced, the grain number of the main stem spike is reduced, and the yield of the single plant is reduced. Therefore, the plant type related protein PROG2 has very important function for regulating and controlling the plant type and the yield of the rice, and has wide prospect in breeding new varieties of high-yield rice.

Drawings

FIG. 1 shows a comparison of the phenotype of Guichao No. 2 and the Dongxiang common wild rice introgression line SIL 40.

FIG. 2 shows the fine localization of the PROG2 gene.

FIG. 3 is T₀The phenotype of the rice plant transformed with the PROG2 gene is compared with that of the middle flower 17.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

Guizhu No. 2 is described in the following documents: zhang X, Zhou S X, Fu Y C, et al.identification of a drop of tall oil interrogue common with wide university (o. rufipogon Griff.). Plant Mol Biol, 2006, 62: 247-259, publicly available from the university of agriculture in china. Gui Dynasty No. 2 is abbreviated as GC2 hereinafter.

Wild rice in east and west of the country is described in the following documents: tian F, Li D J, Fu Q, Zhu Z F, Fu Y C, Wang X K, Sun C Q.2006.construction of the iterative lines carrying with edge (Oryza rufipogon G.F.) segments in collective edge (O.sativa L.) background and conversion of iterative segments associated with edge-related information, clinical and Applied Genetics, 112, 570-80. publicly available from the university of agriculture in China.

The Dongxiang common wild rice introgression line SIL40, which is the progeny of multiple crosses and backcrosses of GC2 with Jiangxi Dongxiang wild rice, is described in the following documents: tian F, Li D J, Fu Q, Zhu Z F, Fu Y C, Wang X K, Sun CQ.2006.construction of the iterative lines carrying with edge (Oryza rufipogon Griff.) segments in summarized edge (O.sativa L.) background and characterization of the iterative segments associated with edge-related information, Theoretical and Applied Genetics, 112, 570-80. publicly available from the university of agriculture in China. The Dongxiang common wild rice introgression line SIL40 is hereinafter abbreviated as SIL 40.

ZH17 is described in the following documents: tan L B, Li X R, Liu F X, Sun X Y, Li C G, Zhu Z F, Fu Y C, Cai H W, Wang X K, Xie D X and Sun C Q.control of key transfer from development to attention in place Nature Genetics,2008,40(11):1360-1364, publicly available from the university of agriculture in China.

Agrobacterium tumefaciens EHA105 (named Agrobacterium tumefaciens strain EHA105 in the literature) in the following examples is described in the following documents: GLUTELIN PRECURSOR ACCUMULATION3 codes a regulator of post-Golgi vehicular traffic for vacuolar protein conditioning in rice end patent cell.2014 Jan; 26(1):410-25. publicly available from the university of agriculture in China to repeat the experiments of the present application.

Example 1 obtaining and identification of transgenic Rice with altered plant type and/or altered yield

Discovery of PROG2 Gene

The phenotypes of GC2 and SIL40 were compared and counted, and the experimental results are shown in fig. 1(a is the overall plant type comparison at the tillering stage, b is the overall plant type comparison at the filling stage, c is the main stalk spike comparison, d is the tillering number statistical result, e is the tillering angle statistical result, f is the primary stalk number comparison, g is the secondary stalk number comparison, h is the main stalk spike grain number comparison, i is the individual plant yield comparison, wherein P <0.05 difference is significant, and P <0.01 difference is very significant). The results showed that SIL40 grew obliquely, tillering number increased, tillering angle increased, primary stem number decreased, secondary stem number decreased, main stem grain number decreased, and yield per plant decreased, compared to GC 2.

SIL40 was backcrossed to the recurrent parent GC2 to construct a secondary segregating population. The PROG2 gene (shown in sequence 1 of the sequence table) is preliminarily positioned between the short arm SW1 of the rice 7 th chromosome and the ID52 marker by utilizing a secondary segregation population. Further, using a 4000 recessive homozygous individual (upright growth), the PROG2 gene was finely localized between the two markers F43 and ID52 (see fig. 2), with an actual physical distance of 9.2 kb. The open reading frame of the PROG2 gene is shown as 125 th-691 th site from 5' end of sequence 1 in the sequence table, the coded protein is named as PROG2, the amino acid sequence of the protein is shown as sequence 2 in the sequence table, and the protein consists of 188 amino acid residues.

Second, PROG2 gene function verification

1. Construction of recombinant vectors and recombinant Agrobacterium

(1) Taking 2 weeks seedlings of Jiangxi Dongxiang wild rice as experimental materials, extracting genome DNA as a template, and adopting a primer D81-F: 5' -CGGAATTCAAGCTTTGGAGAATAAGTCT-3' (restriction enzyme EcoRI recognition site underlined) and primer D81-R: 5' -TCCCCCGGGTCTAGAAAGGGATTTATCCC-3' (restriction enzyme SmaI recognition site is underlined) to obtain a PCR product of about 4200 bp.

(2) And (3) double-digesting the PCR product obtained in the step (1) by using restriction enzymes EcoR I and Sma I, and recovering a digested product.

(3) The plant expression vector pCAMBIA1300 was digested with restriction enzymes EcoR I and Sma I, and the vector backbone of about 8.9kb was recovered.

(4) And (3) connecting the enzyme digestion product in the step (2) with the vector skeleton in the step (3) to obtain a recombinant plasmid D81.

According to the sequencing result, the recombinant plasmid D81 obtained in step (4) was structurally described as follows: the insertion of a DNA molecule with a nucleotide sequence shown as a sequence 3 in a sequence table between the EcoR I enzyme cutting sites and the Sma I enzyme cutting sites of the plant expression vector pCAMBIA 1300. The recombinant plasmid D81 expresses PROG2 protein shown in a sequence 2 in a sequence table.

the recombinant plasmid D81 contains an expression cassette which comprises a PROG2 promoter, a PROG2 protein coding gene and a PROG2 terminator; the PROG2 promoter is shown as 1 st to 1258 th from 5 ' end of the sequence 3, the gene coding for the PROG2 protein is shown as 1383 th to 1949 th from 5 ' end of the sequence 3, and the PROG2 terminator is shown as 2151 th to 4214 th from 5 ' end of the sequence 3.

The recombinant plasmid D81 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium EHA105/D81 containing recombinant plasmid D81.

The empty vector plasmid pCAMBIA1300 is transferred into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium EHA105/pCAMBIA1300 containing the plasmid pCAMBIA 1300.

2. Acquisition of PROG2 transgenic rice

(1) Seeds of rice variety ZH17 were dehulled and sterilized, then processed by the method of Hiei et al (Hiei Y, Ohta S, Komari T)&Transformation of ZH17 with recombinant Agrobacterium EHA105/D81 resulted in T.origin transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the bases of the T-DNA plant J.1994,6: 271-282)₀The transgenic rice plant with PROG2 gene is substituted.

(2) Separately extracting T₀The genome DNA of a rice plant with the PROG2 gene is substituted and used as a template, and a primer HPT-F: 5 '-TACTTCTACACAGCCATC-3' and primer HPT-R: 5 '-CGTCTGTCGAGAAGTTTC-3', if 947bp amplification product is obtained, the T₀The rice plant with the gene of PROG2 is transformed into T₀Transferring a positive plant of the PROG2 gene.

According to the methods (1) and (2) above, 56T strains were finally obtained₀Transferring a positive plant of the PROG2 gene. Randomly select 3T₀The lines transformed with the positive plant of PROG2 gene were named D81-1, D81-2 and D81-3, respectively, and the following experiments were performed.

Replacing the recombinant Agrobacterium EHA105/D81 with the recombinant Agrobacterium EHA105/pCAMBIA1300 according to the above method, and obtaining T by the same steps₀Transferring empty carrier positive plants.

3. Phenotype identification of positive plant transformed with PROG2 gene

The rice varieties ZH17 and T₀The positive plant with the transferred empty carrier and the seeds of D81-1 are planted in pots filled with nutrient soil and vermiculite in the volume ratio of 1:1, and are cultured alternately at 25 deg.c under illumination, and the phenotype of rice plant is compared and counted during growth and development. The experiment was repeated three times, 30 replicates each time.

d81-1 is replaced by D81-2 and D81-3 respectively according to the steps, and other steps are the same, so that the statistical result of the phenotype of each rice plant is obtained.

The experimental results are shown in FIG. 3(a is the comparison of the whole plant type in the tillering stage, b is the comparison of the whole plant type in the grain filling stage, c is the comparison of the main stem and spike, d is the statistical result of the plant height, and e is the statistical result of the tillering numberAs a result, f is a comparison of the number of primary shoots, g is a comparison of the number of secondary shoots, h is a comparison of the number of grains of the stem ear, and i is a comparison of the yield per plant, where<0.05 difference was significant, representing P<0.01 difference was very significant). The results show that T₀The transgenic PROG2 positive plants show SIL 40-like phenotype; in contrast to ZH17, T₀The plant height of the transgenic PROG2 gene positive plant is reduced by about one fourth, the tillering number is increased by about one time, the tillering angle is increased, the number of primary branches is reduced by about one fourth, the number of secondary branches is reduced by about two thirds, the number of main stem spikes is reduced by about one half, and the yield of a single plant is reduced by about one third. T is₀the plant height, tillering number, primary branch number, secondary branch number, main stem grain number and single plant yield of the dummy empty vector positive plant and ZH17 have no obvious difference.

The result shows that the PROG2 gene has very important function for regulating the plant type and the yield of the rice.

Claims

1. The amino acid sequence of the protein is shown as a sequence 2 in a sequence table.

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is (b1) or (b2) as follows:

(b1) DNA molecule shown in sequence 1 in the sequence table;

(b2) DNA molecules of 125 th-691 th nucleotides from 5' tail end of a sequence 1 in a sequence table.

4. An expression cassette, recombinant vector or recombinant microorganism comprising the nucleic acid molecule of claim 2 or 3.

5, c1) or c 2):

c1) Use of a protein according to claim 1, or a nucleic acid molecule according to claim 2 or 3, or an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising a nucleic acid molecule according to claim 2 or 3, for modulating plant type and/or yield in a plant;

c2) use of a protein according to claim 1, or a nucleic acid molecule according to claim 2 or 3, or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line comprising a nucleic acid molecule according to claim 2 or 3, for the production of transgenic plants with altered plant type and/or altered yield;

The plant is rice.

6. The use of claim 5, wherein: in the (c2), the yield was changed to a decrease in the yield of the individual plant.

7. The use of claim 5, wherein: in the step (c2), the plant type is changed to a reduced plant height and/or an increased tiller number and/or an increased tiller angle and/or a reduced number of primary shoots and/or a reduced number of secondary shoots and/or a reduced number of main stem ears.

8. Use according to any one of claims 5 to 7, wherein: the rice is rice variety ZH 17.

9. A method of producing a transgenic plant comprising the step of introducing the nucleic acid molecule of claim 2 or 3 into a recipient plant to produce a transgenic plant; compared with the acceptor plant, the transgenic plant has the advantages that the yield per plant is reduced, the plant height is reduced, the tillering number is increased, the tillering angle is increased, the number of primary branches is reduced, the number of secondary branches is reduced, and/or the number of main stem ears is reduced; the plant is rice.

10. The method of claim 9, wherein: the rice is rice variety ZH 17.