WO2021114156A1 - Petal purple spot protein and coding gene thereof - Google Patents

Abstract

Provided is a petal purple spot protein, the protein comprising the amino acid sequence shown in SEQ ID No. 1. Also provided is a gene encoding a protein comprising the nucleotide sequence shown in SEQ ID No. 2. Further provided is an application of the protein and the coding gene thereof in regulating purple spot traits of plant petals. The present invention confirms that the purple spot trait in petals is controlled by a single dominant gene. The purple spot trait in petals can be used as a marker trait for sea-land hybrids. The gene can also be transferred into floral plants by transgenic methods, providing a novel gene source for expanding the variety of floral plants.

Description

Petal purpurin and its coding gene Technical field

The invention belongs to the technical field of plant genetic engineering, and in particular relates to a petal purpurin; and also relates to a gene encoding the petal purpurin and its application.

Background technique

The heterochromatic spots and stripes on the flowers, leaves, fruits and branches of plants are collectively called colorful spots, especially the colorful spots on the petals, which constitute the main component of the ornamental value of horticultural plants. According to the size, shape and position of the petals, the colorful spots on the petals are divided into two categories: regular colorful spots and irregular colorful spots. Regular colorful spots include garland, flower center (flower eye), flower spot, flower rib and There are many forms such as lace; irregular color spots refer to heterochromatic scattered dots or stripes with non-fixed patterns on the petals. Regular colored spots are usually controlled by genes and can be passed on in accordance with hereditary rules during sexual crossing.

Cotton is an important fiber cash crop. Upland cotton (Gossypium hirsutum L.) and sea island cotton (Gossypium barbadense L.) are two main cultivated cottons. Although both are heterotetraploid cotton species formed by interspecific hybridization between diploid Asian cotton (A genome) and Raymond cotton (D genome), they were formed during natural selection and artificial domestication. The difference in chromosome structure causes significant differences in morphological characteristics, fiber quality, and adaptability between upland cotton and sea island cotton. For example, there are purple spots (purple spots for short) at the base of the petals of sea island cotton varieties, while most upland cotton varieties do not have purple spots at the base of the petals.

At present, the genetic regulation of the purple spot of the sea island cotton petal is still unclear.

Summary of the invention

The inventors discovered that the purple spot at the base of the petals of the sea island cotton is controlled by a single dominant gene.

In order to take advantage of the purple spot characteristics of the petal base of the sea island cotton, the present invention provides a petal purple spot protein named GhBM. The protein is the following protein (1), (2) or (3):

(1) A protein consisting of the amino acid sequence shown in SEQ ID No: 2;

(2) The amino acid sequence shown in SEQ ID No: 2 is a protein obtained by substitution, and/or deletion, and/or addition of one or several amino acid residues, and having the same function.

(3) A protein that has more than 90% identity with the amino acid sequence shown in SEQ ID No: 2 and has the same function.

The present invention also provides the application of the above-mentioned protein in regulating and controlling purple spot of plant petals.

The plant refers to cotton or floral plants and the like.

The present invention also provides a gene encoding the petal purpurin, named GhBM, and the gene is a nucleic acid molecule as described in (a), (b), (c) or (d) as follows:

(a) Consists of the nucleotide sequence shown in SEQ ID No: 1;

(b) A nucleotide sequence that has more than 90% identity with the nucleotide sequence shown in SEQ ID No: 1 and encodes the same functional protein;

(c) A nucleotide sequence that has more than 95% identity with the nucleotide sequence shown in SEQ ID No: 1 and encodes the same functional protein;

(d) A nucleotide sequence that hybridizes to the nucleotide sequence defined in (a), (b) or (c) under stringent conditions and encodes the same functional protein.

The present invention also provides a recombinant expression vector containing the above gene.

The expression vector refers to plant expression vectors such as pBI121, pCAMBIA1300, and pCAMBIA2300.

The present invention also provides microorganisms, cell lines or plants containing the above-mentioned genes or recombinant expression vectors.

The present invention also provides the application of the above-mentioned gene or recombinant expression vector in regulating and controlling purple spot of plant petals.

The plant refers to cotton or floral plants and the like.

The flower plant refers to gladiolus (Gladiolus gandavensis, also known as gladiolus), rose (Rosa rugosa Thunb.), carnation (Dianthus caryophyllus), dianthus (Dianthus chinensis L.), narcissus (Narcissus tazetta L. var. chinensis) Roem), rose (Rosa chinensis Jacq), tulip (Tulipa gesneriana L.), magnolia (Magnolia denudata Desr), peony (Paeonia suffruticosa Andr.), peony (Paeonia lactiflora Pall.), lilac (Syringa (Cinn)), Bauhinia chinensis), Cyclamen (Cyclamen persicum Mill.), Hyacinth (Hyacinthus Orientalis L.), Calla lily (Zantedeschia aethiopica (L.) Spreng.), Perennial chrysanthemum, Geranium (Pelargonium hortorum), Primula (Primula Ranchalacoides) .), Cineraria hybrida (Pericallis hybrida), Petunia hybrida (Petunia hybrida), Poppy (Papaver rhoeas L.), Snapdragon (Antirrhinum majus L.), Hibiscus (Hibiscus rosa-sinensis), Hibiscus (Hibiscus mutabilis) Linn. ), oleander (Nerium indicum Mill.), dahlia (Dahlia pinnata Cav.), five-color plum (Lantana camara L.), canna (Canna indica L.), sunflower (Helianthus annuus L.), a bunch of red (Salvia) splendens Ker-Gawler, Celosia cristata L., Impatiens balsamina L., Scutellaria barbata D. Don, Amaranthus tricolor, Dendrathema morifolium (Ramat. ) Tzvel.), lotus (Nelumbo SP.) or water lily (Nymphaea L.), etc.

The present invention also provides a method for cultivating plant varieties with purple spots on petals, which comprises introducing the above-mentioned genes into target plants to obtain transgenic plants with purple spots on petals.

The target plants mentioned in the above methods refer to cotton or floral plants and the like.

The present invention has the advantages and beneficial effects as follows: (1). The present invention provides single dominant gene control for the purple spot basal traits at the base of the sea island cotton petals. It also accurately locates the gene and obtains the nucleotide of the gene. The sequence provides a new source of genes for the gene transfer into flower plants through transgenic methods, and for the colorfulness of flower plants. (2) The present invention clarifies that the purple spot traits at the base of the island cotton petals are controlled by a single dominant gene, which can be used as a marker shape for rapid identification and screening of sea-land hybrids and as a marker trait for identifying the purity of sea-land hybrids.

Description of the drawings

Figure 1 shows the phenotypic photos of the floral organs of the sea island cotton HaiR and the upland cotton P30A; among them, 1 is the upland cotton variety P30A, and B is the sea island cotton variety HaiR.

Figure 2 is the electrophoresis map of GhBM gene linkage marker screening; among them, 1 is the PCR amplification result of upland cotton P30A primer A07INS8; 2 is the PCR amplification result of sea island cotton HaiR primer A07INS8; 3 is the _{mixed pool of 24 F 2} single plants without purple spots The PCR amplification result of primer A07INS8; 4 is the PCR amplification result of upland cotton P30A primer A07INS13; 5 is the PCR amplification result of sea island cotton HaiR primer A07INS13; 6 is the primer A07INS13 of 24 F ₂ generation single plant mixed pool without purple spot PCR amplification results.

Figure 3 is a schematic diagram of PCR amplification of GhBM gene linkage marker in F ₂ generation population without purple spots; among them, 1 is the PCR amplification result of the HaiR primer A07DEL9 of sea island cotton; 2 is the PCR amplification result of the upland cotton P30A primer A07DEL9; 3-25 are The PCR amplification results of the primer A07DEL9 of 23 F _{2 generation single plants without purple spots.}

Figure 4 is the electrophoresis map of the GhBM gene linkage marker in the F ₂ generation population without purple spots; among them, 1 is the PCR amplification result of the HaiR primer A07DEL11 of sea island cotton; 2 is the PCR amplification result of the upland cotton P30A primer A07DEL11; 3-25 are 23 strains The _{results of PCR amplification of primer A07DEL11 of F 2} mixed pool of single plant without purple spots.

Figure 5 is a photo of flower bud size at different sampling periods; 1 is the young bud period; 2 is the flower bud period; 3 is the flowering period.

Figure 6 is a histogram of the expression levels of GhBM gene in HaiR and P30A at different stages and different parts of petals; among them, 1 is the purple spot area at the young bud stage of HaiR, 2 is the purple spot area at the young bud stage of HaiR; 3 is the purple spot area at the flower bud stage of HaiR ; 4 is the area with no purple spots at the flower bud stage of HaiR; 5 is the area with purple spots at the flowering stage of HaiR; 6 is the area without purple spots at the flowering stage of HaiR; 7 is the area with purple spots at the young bud stage of P30A; 8 is the area without purple spots at the young bud stage of P30A; 9 is the area without purple spots at the young bud stage of P30A. Purple spot area during the P30A bud stage; 10 is the P30A flower bud stage without purple spot; 11 is the P30A bloom stage purple spot area; 12 is the P30A bloom stage without purple spot area;

Figure 7 is a bar graph of fluorescence quantitative PCR detection of GhBM gene expression in sea island cotton HaiR that inhibits GhBM gene; where 1 is HaiR-CK; 2 is HaiR-BMKO1; 3 is HaiR-BMKO2; 4 is HaiR-BMKO3.

Figure 8. The phenotype of the young bud petals of the GhBM gene suppressed in the sea island cotton HaiR; 1 is HaiR-CK; 2 is HaiR-BMKO.

Figure 9 is a phenotype photograph of the petals at the flowering stage of the GhBM gene suppressed in the sea island cotton HaiR; 1 is HaiR-CK; 2 is HaiR-BMKO.

Detailed ways

The following examples further illustrate the present invention, but do not limit the protection scope of the present invention.

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

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The TRV-00 vector and Agrobacterium GV3101 are described in the article "Liang, C et al. (2016) GhABF2, abZIP transcription factor, confers drought and salary tolerance in cotton (Gossypium hirsutum L.). Sci Rep, 6,35040.".

Example 1. Genetic analysis of the purple spot traits at the base of the petals of sea island cotton

(1) Test materials: Upland cotton variety P30A, there is no purple variegation at the base of the petal (hereinafter referred to as "petal purple patch") (see Figure 1); the sea island cotton variety HaiR (formerly known as ISR, the applicant self-bred island Cotton variety) (see Figure 1), with purple mottling at the base of the petals.

(2) Test method:

Crossing the upland cotton variety P30A as the female parent and the sea island cotton variety HaiR as the male parent, the hybrid F ₁ generation was obtained; then the F ₁ generation was selfed to obtain the F ₂ generation; in the flowering period, the F ₁ generation and F _{2 were investigated and counted} The number of individual plants with purple variegation and no purple variegation at the base of the petals.

Results _{All plants of F 1} generation had purple spots at the base of petals, indicating that the characteristics of petal purple spots were controlled by dominant genes. A total of 5540 individual plants were investigated in the F ₂ generation, and the results (see Table 1) The number of individual plants with purple spots at the base of the petals was 4255, and the number of individual plants without purple spots at the base of the petals was 1285. The proportion of individual plants with purple spots: no purple spots was about 3:1, indicating that the petal purple spot is controlled by a single dominant gene. The gene that controls the purple spots at the base of the petals is named: GhBM.

Table 1 Segregation ratio of purple spot traits at the base of petals in _{F 2} generations of cross between sea island cotton and upland cotton

Example 2 Map-based cloning of cotton petal purple spot GhBM gene

(1) Test materials: _{5540 strains of the F 2} generation segregated population obtained in Example 1.

(2) Test method:

(1) DNA extraction, using DNAsecure Plant Kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) and extracting genomic DNA according to the method provided in its manual.

(2) Preliminary localization of GhBM gene: The _{mixed pool of 48 cotton genomic DNA without petal purple spots in the F 2} segregated population was used as a template, and 720 pairs of polymorphic primers between upland cotton and sea island cotton developed by the applicant were used as primers Carry out PCR amplification for preliminary localization of the GhBM gene.

The above PCR amplification: the polymorphic primers used (see Table 2), using 2×Taq PCR StarMix with Loading Dye reagent (purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd.) for PCR amplification.

The PCR reaction system (20 μl) is: 1 μl of genomic DNA, 10 μl of PCR Mix, 1 μl of upstream primer, 1 μl of downstream primer, and 7 μl of ddH ₂ O. The PCR reaction program is: 94°C for 10 minutes; 94°C for 15 seconds, 15 seconds according to primer annealing temperature, 72°C for 1 minute, 35 cycles; 72°C for 10 minutes, 16°C hold. The obtained PCR amplification products were detected by 1% agarose gel electrophoresis.

The results (see Figure 2) primers A07INS8 and A07INS13 of A07 chromosome were significantly linked to the petal purple spot. This indicates that the gene is located on chromosome A07.

(3) Fine mapping of GhBM gene

Download the whole genome resequencing (WGS) data of sea island cotton released by Nanjing Agricultural University from the SRA database of NCBI (https://www.ncbi.nlm.nih.gov/sra), and use NGSQCToolkit to filter the downloaded WGS data , Exclude the low-quality sequence, use BWA software to compare the sequencing of sea island cotton to the upland cotton genome (upland cotton genome published by Nanjing Agricultural University), use Pindel and Samtools software to find the fragment length between sea island cotton and upland cotton For InDel sites with a difference of less than 80bp, primers were designed on both sides of each polymorphic site, and TNT blast was used to detect the matching of the primers on the genome (NAU). The designed polymorphic primers were used to carry out PCR amplification on 1285 single plants without purple spots in the _{F 2 generation population (sea and land), and further fine positioning was performed.}

The above-mentioned PCR amplification: the polymorphic primers used (see Table 2), using 2×Taq PCR StarMix with Loading Dye reagent (purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd.) for PCR amplification.

The PCR reaction system is: DNA 1 μl, PCR Mix 10 μl, primer F 1 μl, primer R 1 μl, ddH ₂ O 7 μl, and a total volume of 20 μl. The PCR reaction program is: 94°C for 10 minutes; 94°C for 15 seconds, 15 seconds according to primer annealing temperature setting, 72°C for 1 minute, 35 cycles; 72°C for 10 minutes, kept at 16°C. The obtained PCR amplification products were detected by 1% agarose gel electrophoresis.

Table 2 Primer sequences involved in map-based cloning

Table 3 Map-based cloning of 7 gene annotations within 70kb

Gene number	Gene name	Gene description
G1	bHLH	Putative transcription factor bHLH
G2	NA	unknown
G3	HMGS	Hydroxymethylglutaryl-CoA synthase
G4	CML	Possible Calbindin CML
G5	At1g	Possible fructokinase-6, chloroplast
G6	CML	Possible Calbindin CML
G7	MYB	Transcription factor MYB

Results: Further PCR amplification analysis of a single plant showed that the gene controlling the purple spot at the base of the sea island cotton petals was located between the two polymorphic primers A07INS8 and A07INS13 (see Table 2 for primer sequences). By expanding the _{number of F 2} populations, we developed CAPS (Cleaved amplified polymorphic sequences) primers (see Table 2 for primer sequences), and further fine-tuned the positioning. Through map-based cloning, the control petal base purple spot gene was mapped to the two primers A07INS10.3 and A07Del10.1 Within the range of 70kb (see Figure 3 and Figure 4). According to the annotation results of CottonFGD (www.cottonfgd.org/), there are 7 genes in this range (see Table 3), which are named G1-G7 in turn. Among them, the G7 gene encodes an R2R3MYB transcription factor. In other species, R2R3MYB transcription factors regulate the color formation of plant flowers, fruits, leaves, etc. (Cao X et al, J Exp Bot, 2017, 68, 5745-5758). Therefore, It is speculated that G7 gene is the most likely candidate gene to regulate petal purpura. Comparing and analyzing with the software of Clutal W, it is found that there is a SNP difference in the coding region of G7 gene between Gossypium hirsutum and Gossypium hirsutum, which causes an amino acid residue of the encoded protein to change from aspartic acid (Asp, D) to alanine (Ala ,A).

Example 3 Analysis of GhBM gene expression in cotton petal purplish spot

(1) Test materials: Flowers of upland cotton P30A and sea island cotton HaiR at different developmental stages, and different parts of the petals.

(2) Test method:

1. RNA extraction:

EASYspin plant ultrapure RNA rapid extraction kit (purchased from Yuan Pinghao (Beijing) Biotechnology Co., Ltd.) was used for RNA extraction. The specific operations are as follows:

a) Add 500μl of Lysis Buffer RLT (with β-mercaptoethanol) to a 2ml sterile RNA centrifuge tube, then add 50μl PLANTaid, and mix well for later use;

b) Take 100mg of cotton P30A or HaiR petal tissue, grind it into powder with liquid nitrogen, add 550μl of the prepared lysis solution, immediately vortex for 1min, and let it stand for 10min at room temperature to make it fully lysed;

c) Centrifuge at 4°C at 12000rpm for 10min, and transfer 500μl of supernatant to a new 1.5ml RNase-free centrifuge tube;

d) Add 0.5 times volume of absolute ethanol (250μl) and mix;

e) Add the mixture to the adsorption column RA, centrifuge at 12000 rpm at 4°C for 1 min, and discard the waste liquid;

f) Add 2μl RNase inhibitor+5μl DNase+20μl DNA Buffer, let stand at room temperature for 10 minutes, add 700μl protein-removing solution RW1, leave it at room temperature for 30s, centrifuge at 4℃, 12000rpm for 1min, discard the waste liquid;

g) Add 500μl of rinsing solution RW (make sure to add absolute ethanol beforehand), centrifuge at 4°C, 12000rpm for 1min, and discard the waste solution. Repeat washing once;

h) Put the adsorption column RA back into the collection tube at 4°C, 12000rpm for 2 minutes, take out the adsorption column, put it into a new 1.5ml RNase-free centrifuge tube, and dry in the ultra-clean table until there is no alcohol residue (5min) ；

i) Add 30μl RNase-free water (preheated at 65°C) dropwise to the middle of the adsorption membrane of the adsorption column, leave it at room temperature for 2 minutes, and centrifuge at 4°C for 2 minutes at 12000 rpm. (Take 2μl RNA and use a nano instrument to detect RNA concentration).

(2) Reverse transcription to cDNA

Reverse transcription system (20μl): (RNA 3μl, Oligo dT 1μl, RNase-free water 4μl) Preheat at 65°C for 5min, TransScript RT/R1 enzyme mix 1μl, gDNA Remove 1μl, 2×TS Reaction Mix 10μl.

PCR reaction program: 42°C for 15min, 85°C for 5s.

(3) Real-time fluorescent quantitative PCR (qRT-PCR)

The specific detection method, data processing, internal reference primer sequence and petal purple spot gene detection primer sequence are as follows:

According to the usage method provided by Bio-Rad, SYBR green Ⅰ fluorescent dye was added to the PCR system, and the ^{G1-G7 gene was detected on the fluorescent quantitative PCR instrument C1000 TM} Thermal Cycler (CFX96 real-time system, Bio-Rad, USA) Expression situation: The cotton ACTIN7 gene was used as the internal control, and the experiment was set for three repetitions. The primer sequence is as follows.

Among them, the primer sequence for amplifying the internal reference ACTIN7 is:

ACTIN7 upstream primer: 5'-CTCTCTCTGTATGCCAGTGGTC-3';

ACTIN7 downstream primer: 5'-TTGTCCGTCAGGCAACTCATAG-3'.

The primer sequence for amplifying GhBM is:

GhBM gene upstream primer: 5'-ACGAACAGCAAACGATGTGA-3' (SEQ ID No. 3);

Downstream primer of GhBM gene: 5'-CTATTGTTGCCGCTGTCAGA-3' (SEQ ID No. 4).

Data processing adopts the method of Comparative Ct, that is, the Ct value is the number of cycles experienced when the fluorescent signal in the PCR tube reaches the set threshold, ΔCt=Ct(SAGs)-Ct(ACTIN1), and the 2- ^ΔCt value is used to measure gene transcription Level, analysis of GhBM gene expression in sea island cotton and upland cotton

The PCR reaction system (20 μl) is: cDNA 1 μl, KOD SYBR qPCR MIX 10 μl, primer F 0.5 μl, primer R 0.5 μl, and ddH ₂ O 8 μl. The PCR reaction program is: 98°C for 2min; 98°C for 10s, 60°C for 10s, 68°C for 30s, 40 cycles; 95°C for 15s, 60°C for 15s, 95°C for 10s.

Results (see Figure 6) Compared with upland cotton P30A, the expression of GhBM gene in the purplish spots at the base of the sea-island cotton HaiR petals was significantly higher, while the expression in the non-purple spots was relatively low; while in P30A, the expression of GhBM in different parts of different flowering stages The amount is low, or even undetectable, indicating that the GhBM gene is a gene that regulates the formation of purple spots at the base of the island cotton petals. That is, the increase of GhBM gene expression in the base of the HaiR petals of sea island cotton led to the formation of purple spots.

Example 4. GhBM gene interference expression test

(1) Test materials: Sea island cotton variety HaiR, VIGS interference expression vector and Agrobacterium strain GV3101 (preserved by the applicant's laboratory).

(2) Test method:

(1) Refer to the method described in Example 3 for the extraction of total RNA of sea island cotton HaiR and the acquisition of cDNA.

(2) Construction of recombinant expression vector VIGs-GhBM

(a) Primer design: Find a specific segment in the coding region of the GhBM gene to design primers VRB-F and VRB-R. The primer sequences are:

VRB-F: 5'-gtca ggatcc AAGGGAAATGGCATCAAGTG-3' (SEQ ID No. 5) (the underlined and italicized part is the recognition site of BamHI);

VRB-R: 5'- ggtacc gacCGGGTTCGAAGTTTTACTGG-3' (SEQ ID No. 6) (underlined and italicized is the recognition site of Kpn I).

(b) PCR amplification: using the cDNA obtained in step (1) as a template, and VRB-F and VRB-R as primers for PCR amplification, a DNA fragment of 348 bp is obtained, and the PCR product obtained is purified and sequenced. The obtained fragment is The DNA fragment in sequence 1 of the sequence listing.

(c) Double digestion of the PCR product obtained in (b) with BamHI and Kpn I, recover the digested fragments, and connect them with the vector TRV-00 backbone fragment that has been digested with BamHI and Kpn I to obtain a recombinant plasmid. The VIGS interference expression vector (TRVGhBM vector). The recombinant plasmid was sent for sequencing, and the sequencing showed that the DNA fragment in the sequence table was inserted between the restriction sites BamHI and Kpn I of the TRV vector, and the recombinant plasmid was named TRVGhBM. After infecting cotton leaves, viruses carrying TRVGhBM gene fragments can induce the silencing of cotton GhBM genes, thereby affecting its biological function of regulating the development of purple spots at the base of petals.

The construction of the aforementioned recombinant plasmid TRVGhBM can also use the artificially synthesized petal purple spot gene shown in sequence 1 of the sequence table as a template.

(3) The VIGS system of GhBM gene interferes with the acquisition of cotton

The recombinant plasmid TRVGhBM constructed in step (2) was introduced into the sea island cotton variety HaiR through Agrobacterium GV3101. For specific transformation and screening methods, see "Liang, C et al. Sci Rep, 2016. (6), 35040.". At the same time, a control of TRV-00 and TRVGbCLA1 transferred into the empty carrier (gifted by the laboratory of Professor Zhang Xianlong of Huazhong Agricultural University) was set up. Finally, three transiently expressed transgenic cotton plants were obtained, namely the sea island cotton plants transformed into the TRVGhBM vector, collectively referred to as HaiR-BMKO, and the 3 plants obtained were specifically named HaiR-BMKO1, HaiR-BMKO2 and HaiR-BMKO3; those transformed into TRVGbCLA1 The sea island cotton plant was named HaiR-CLAKO; the sea island cotton plant transferred into the TRV-00 empty carrier was named HaiR-CK.

(4) GhBM expression analysis in TRVGhBM cotton

Collect the cotton TRVGhBM material petals obtained in step (3), and analyze the expression of GhBM in the TRVGhBM cotton HaiR-BMKO according to the RNA extraction, reverse transcription, qRT-PCR and other methods in Example 3. The control group HaiR-CK .

Quantitative fluorescent PCR results (see Figure 7), compared with HaiR-CK, the expression of GhBM gene in HaiR-BMKO cotton was significantly reduced, indicating that the experiment of interference gene expression was successful.

(5) Phenotype analysis of TRV GbCLA1 in the transgenic control group

Two weeks after the injection, the color change of the new leaves of TRV-CLAKO cotton obtained in step (3) was observed. The results showed that the new leaves of TRV-CLAKO cotton showed obvious albino or spot-like albino, indicating that the VIGS infection event was successful, and neither the strain nor the carrier was successful. There is a problem. The HaiR-CK transferred to TRV-00 with no load is the experimental control. Observe the flowers of the cotton HaiR-BMKO interference line obtained in step (3), and observe the changes of the purple spots at the base of the petals of each interference cotton line, including the presence or absence, size and For changes in color shades, at least 20 flowers should be observed for each transgenic event.

Results: Compared with HaiR-CK, HaiR-BMKO cotton petal base development was not affected in any way. However, in HaiR-BMKO interference cotton lines, the development of purple spots at the petal base at the young bud stage was significantly inhibited or even disappeared (see Figure 8); During the period, compared with the control group HaiR-CK, HaiR-BMKO did not observe the appearance of purple spots (see Figure 9). It shows that the GhBM gene is a gene that controls the formation of purple spots at the base of the petals.

Claims (12)

1. A petal purpura protein, characterized in that the protein is the following (1), (2) or (3) protein:

   (1) A protein consisting of the amino acid sequence shown in SEQ ID No: 2;

   (2) The amino acid sequence shown in SEQ ID No: 2 is a protein obtained by substitution, and/or deletion, and/or addition of one or several amino acid residues, and having the same function.

   (3) A protein that has more than 90% identity with the amino acid sequence shown in SEQ ID No: 2 and has the same function.
2. The application of the petal purpura protein of claim 1 in regulating and controlling plant petal purpura.
3. The application according to claim 2, wherein the plant is cotton or floral plant.
4. The gene encoding the petal purpurin of claim 1, wherein the gene is a nucleic acid molecule as described in (a), (b), (c) or (d) below:

   (a) Consists of the nucleotide sequence shown in SEQ ID No: 1;

   (b) A nucleotide sequence that has more than 90% identity with the nucleotide sequence shown in SEQ ID No: 1 and encodes the same functional protein;

   (c) A nucleotide sequence that has more than 95% identity with the nucleotide sequence shown in SEQ ID No: 1 and encodes the same functional protein;

   (d) A nucleotide sequence that hybridizes to the nucleotide sequence defined in (a), (b) or (c) under stringent conditions and encodes the same functional protein.
5. A recombinant expression vector containing the gene of claim 4.
6. The recombinant expression vector according to claim 5, wherein the vector is pBI121, pCAMBIA1300 or pCAMBIA2300.
7. A microorganism, cell line or plant containing the gene of claim 4 or the recombinant expression vector of claim 7.
8. Application of the gene of claim 4 or the recombinant expression vector of claim 5 in regulating the purple spot of plant petals.
9. The application according to claim 8, characterized in that the plant is cotton or floral plant.
10. The application according to claim 3 or 9, characterized in that, the floral plant refers to gladiolus (Gladiolus gandavensis), rose (Rosa rugosa Thunb.), carnation (Dianthus caryophyllus), dianthus (Dianthus chinensis L.) , Narcissus (Narcissus tazetta L.var.chinensis Roem), Rose (Rosa chinensis Jacq), Tulip (Tulipa gesneriana L.), Magnolia (Magnolia denudata Desr), Peony (Paeonia suffruticosa Andr.), Paeonia (Paeonia Pall.flora) , Lilac (Syringa Linn), bauhinia (Cercis chinensis), cyclamen (Cyclamen persicum Mill.), hyacinth (Hyacinthus orientalis L.), calla (Zantedeschia aethiopica (L.) Spreng.), periwinkle (L. Pelargonium hortorum), Primula (Primula malacoides Franch.), Cineraria (Pericallis hybrida), Petunia (Petunia hybrida), Poppies (Papaver rhoeas L.), Snapdragons (Antirrhinum majus L.), Hibiscus (Hibiscus rosa) -sinensis), Hibiscus mutabilis Linn., Nerium indicum Mill., Dahlia pinnata Cav., Lantana camara L., Canna indica L., Sunflower (Helianthus) annuus L., Salvia splendens Ker-Gawler, Celosia cristata L., Impatiens balsamina L., Scutellaria barbata D. Don, Amaranthus tricolor), chrysanthemum (Dendranthema morifolium (Ramat.) Tzvel.), lotus (Nelumbo SP.) or water lily (Nymphaea L.) One of a kind.
11. A method for cultivating plant varieties with purple spots on petals, which is characterized in that it comprises introducing the gene of claim 4 into a target plant to obtain transgenic plants with purple spots on petals.
12. The method according to claim 11, wherein the target plant is cotton or floral plants.

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