CN108949782B

CN108949782B - Gene for controlling colors of sheath and leaf pillow of millet and application thereof in breeding

Info

Publication number: CN108949782B
Application number: CN201810965367.5A
Authority: CN
Inventors: 白辉; 宋振君; 王永芳; 李志勇; 董志平; 全建章; 刘磊; 马继芳
Original assignee: Grain Research Institute of Hebei Academy of Agriculture and Forestry Sciences
Current assignee: Grain Research Institute of Hebei Academy of Agriculture and Forestry Sciences
Priority date: 2018-08-23
Filing date: 2018-08-23
Publication date: 2022-03-22
Anticipated expiration: 2038-08-23
Also published as: CN108949782A

Abstract

A gene for controlling the color of a sheath and a pillow of a millet leaf belongs to the technical field of genetic engineering, and comprises a Ppls1 gene and a Simyb85 gene, wherein the coding sequence of the Ppls1 gene is shown as a sequence table SEQ ID NO: 1, the coding sequence of the Simyb85 gene is shown in a sequence table SEQ ID NO: 2, respectively. The invention also provides the application of the gene for controlling the color of the leaf sheath and the leaf pillow of the millet in millet crossbreeding. The research of the invention is significant in breeding, the determination of the gene of the leaf sheath color of the decampinus farinae Hance of the invention is the reason for determining that the leaf occipital and leaf sheath color of the decampinus farinae Hance is purple in the breeding process, and the gene can be used as a good breeding indicator in breeding, and can intuitively judge whether hybridization succeeds and identify true and false hybrid varieties.

Description

Gene for controlling colors of sheath and leaf pillow of millet and application thereof in breeding

Technical Field

The invention belongs to the technical field of genetic engineering, and relates to a gene for controlling the color of a sheath and a leaf pillow of millet and application thereof in breeding.

Background

A plant of the genus millet of the family Gramineae, which is annual herbaceous, has thick stalks, few tillers, long and narrow tippy leaves, obvious midrib and small vein, and fine hair; panicle-like panicles; the spikelets cluster and grow on the third-level branch stalks, and the spikelets basically have bristles. Every ear has hundreds to thousands of seeds, the seed size is very small, the diameter is about 0.1cm, the ears are generally golden yellow after being matured, the ears are oval, and the grains are mostly yellow. The method is widely cultivated in temperate zones and tropical zones of continental Eurasia, the middle and upstream of the yellow river in China is a main cultivation area, and a small amount of the seeds are cultivated in other areas.

In order to obtain a better millet variety or obtain a millet variety satisfying different traits according to requirements, millet crossbreeding is often required. In the millet crossbreeding process, the identification of whether the progeny is the true hybrid obtained by parent hybridization is a detection work required by breeding workers, however, if the characters of the millet crossbreeding variety are not particularly obvious, it is difficult to visually judge whether the crossbreeding is the true hybrid obtained by parent hybridization, the overall performance, character separation observation or gene identification are often required, and the operation is very troublesome, so that a simple method for identifying true and false hybridization is urgently needed.

Disclosure of Invention

The invention aims to provide a gene for controlling the color of a sheath and a pillow of millet leaves and application thereof. Whether the hybridization is successful or not is judged quickly and intuitively by observing whether the leaf sheath and the leaf pillow are purple or not.

The technical scheme adopted by the invention for realizing the purpose is as follows:

a gene for controlling the color of a sheath and a occipital of a millet comprises a Ppls1 gene and a Simyb85 gene, wherein the coding sequence of the Ppls1 gene is shown as a sequence table SEQ ID NO: 1, the coding sequence of the Simyb85 gene is shown in a sequence table SEQ ID NO: 2, respectively.

A gene for controlling the color of sheath and pillow of millet leaf has the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 by substitution, insertion or deletion, and expressing the same functional protein; or with SEQ ID NO: 1, SEQ ID NO: 2 has more than 90% of homology and expresses the nucleotide sequence or complementary sequence of the same functional protein.

An application of the gene for controlling the color of leaf sheath and leaf pillow of millet in cross breeding of millet.

The CDS coding sequence of the gene is obtained by collecting the purple leaf sheath of the decampine of foxtail millet, extracting mRNA, carrying out reverse transcription to obtain a cDNA sample as a template DNA and carrying out PCR amplification.

The invention has the beneficial effects that: the research of the invention is significant to the breeding aspect, the genes for controlling the color of the leaf sheaths and the leaf pillows of the foxtail millet determined by the invention are the main reasons for determining the purple color of the leaf sheaths and the leaf pillows of the foxtail millet in the breeding process, and the genes can be used as a good breeding indicator in breeding, so that whether hybridization succeeds or not can be intuitively judged, and true and false hybrid varieties can be identified.

The invention is used as a breeding indicator in breeding, has breeding indicating characters, can use a millet variety containing the gene as one of hybridization parents, or constructs the gene on a transgenic carrier and transforms the gene into a target millet, uses the material containing the gene as the parent for hybridization breeding, and can identify true and false hybrids through color phenotypes of filial generations, thereby being convenient and practical.

Drawings

FIG. 1 is a graph showing the expression analysis of two genes, Ppls1 and Simyb85, in Pansy and Yugu No. 1.

FIG. 2 is an analysis chart of the expression of the two genes Ppls1 and Simyb85 in 12 varieties of millet.

FIG. 3 is a diagram of the nucleotide sequence encoding the Ppls1 gene and the corresponding gene of millet No. 1 (Seita.7G195400.1) of the millet reference genome.

FIG. 4 is a diagram showing the amino acid sequence encoded by Ppls1 gene and the corresponding gene of millet No. 1 (Seita.7G195400.1) of the millet reference genome.

FIG. 5 is a diagram of the nucleotide sequence of the gene Simyb85 and the corresponding gene of millet No. 1 (Seita.4G086400.1) of the reference genome of millet.

FIG. 6 is a diagram of the amino acid sequence of the gene Simyb85 and the corresponding gene of millet No. 1 (Seita.4G086400.1) of the millet reference genome.

FIG. 7 is a schematic diagram of a simple model of the synthetic pathway for anthocyanins.

FIG. 8 is an analysis chart of expression of 8 structural genes in the biosynthesis pathway of anthocyanin in Yugu No. 1 and Shilixi.

FIG. 9 is a subcellular localization map of PPLS1 and SiMYB85 proteins.

FIG. 10 is a schematic representation of the full and partially truncated amino acid sequence of PPLS 1.

FIG. 11 is a diagram showing the transcriptional activation analysis of different amino acid sequences of PPLS1 in yeast.

FIG. 12 is a schematic representation of full-length and partially truncated amino acid sequences of SiMYB 85.

FIG. 13 is a graph of the transcriptional activation analysis in yeast of different length amino acid sequences of SiMYB 85.

FIG. 14 is a graph showing the anthocyanin content.

FIG. 15 is a graph of yeast two-hybrid detection of the interaction of PPLS1 and SiMYB85 proteins.

Detailed Description

The research and utilization of the gene have great theoretical significance, which is the important development and update of the traditional polygene hypothesis, the research of the gene provides new resources for the new development of breeding and accelerates the breeding pace, and the invention is further explained by combining with specific embodiments.

Example 1

The expression of the two genes Ppls1 and Simyb85 in Yugu No. 1 of green leaf sheath and Shilixiang of purple leaf sheath is shown in FIG. 1. Wherein L represents the leaf, P represents the leaf pillow, and LS represents the leaf sheath.

Example 2

The expression of the two genes Ppls1 and Simyb85 in 5 parts of green-leaf Anoplophorae semen variety and 7 parts of purple-leaf Anoplophorae semen variety is shown in FIG. 2. Wherein A represents Ppls1, and B represents Simyb 85.

Example 3: sequence difference and protein characteristic analysis of Ppls1 and Simyb85 genes in Yugu No. 1 and Shilixi

Alignment of Ppls1 with the corresponding gene of millet reference genome Yugu No. 1 (Seita.7G195400.1) coding sequence, there were 11 SNPs, 2 insertion mutations of 4bp and 5bp, resulting in 7 amino acid mutations and 3 amino acid insertions, as shown in FIGS. 3-4. PPLS1 encodes a Myogenic helix-loop-helix transcription factor consisting of 571 amino acids, with a theoretical molecular weight of 62.5 kD. The Pfam database (http:// Pfam. xfam. org /) shows that the N-terminus (1-201aa) of the protein has a DNA binding HLH domain (PF00010) belonging to the MYB and MYC transcription factor families.

When the Simyb85 is aligned with the corresponding gene No. 1 of the millet reference genome (Seita.4G086400.1), 2 SNPs and 1 deletion mutation of 3bp exist, so that 2 amino acid mutations and 1 amino acid deletion are caused, as shown in FIGS. 5-6. SiMYB85 belongs to MYB family transcription factor, and encodes a millet anthocyanin consisting of 268 amino acids for regulating C1 protein, and the theoretical molecular weight is 28.4 kD.

Example 4: effect on structural Gene expression in the anthocyanin biosynthesis pathway

To examine the differences in anthocyanin biosynthesis mechanisms between Shiranxiang and Yugu No. 1, we determined the expression of 8 structural genes involved in the anthocyanin biosynthesis pathway, including SiPAL, SiC4H, SiCHS, SiCHI, SiF3H, SiDFR, SiANS and Si3GT, as shown in FIG. 7. Among them, the expression amounts of four genes, SiF3H, SiDFR, SiANS and Si3GT, in the sample of the leaf sheath of purple leaf of Shiranxiang are much higher than those of the sample of the leaf sheath of green leaf of Yugu No. 1 and the leaf of green leaf of Shiranxiang, and the expression patterns are similar to Ppls1 and Simyb85, as shown in FIG. 8. Wherein L represents the leaf, P represents the leaf pillow, and LS represents the leaf sheath.

Example 5: the two proteins PPLS1 and SiMYB85 are located in the nucleus

Nuclear localization is one of the most prominent features of most transcription factors. To examine the subcellular localization of both PPLS1 and SiMYB85 proteins, we fused the fluorescent protein YFP to the C-terminal of PPLS1 and SiMYB85, respectively, to form PPLS1-YFP and SiMYB85-YFP fusion proteins. As a result of observation by confocal laser microscopy, as shown in FIG. 9, PPLS1-YFP and SiMYB85-YFP fusion proteins transiently expressed under the drive of CaMV35S promoter were able to emit green fluorescence in the nucleus of rice protoplast, respectively, whereas the YFP empty vector control green fluorescence signal was distributed throughout the nucleus, cytoplasm and cell membrane of the protoplast cell. These results indicate that PPLS1 and SiMYB85 are both nuclear localization proteins. In FIG. 9, YFP represents the fluorescence signal, mCherry represents the nuclear localization signal, Bright Field represents the superposition of YFP and mCherry in the Bright Field.

Example 6: PPLS1 gene has transcription activation activity, while SiMYB85 has no transcription activation activity

Transcriptional activation is also one of the characteristics of many transcription factors, and we examined the transcriptional activation activity of the genes ppls1 and Simyb85 in yeast.

First, 8 different PPLS1 domains (full-length PPLS1 and partially truncated PPLS1 fragment, in turn designated PPLS1^1-571、PPLS1^1-201、PPLS1^202-377、PPLS1^378-429、PPLS1^430-571、PPLS1^1-377、PPLS1^378-571And PPLS1^202-429FIG. 10) are respectively connected with GAL4-DNA binding structural domain in pGBKT7 vector to construct yeast fusion expression vector, and then the yeast fusion expression vector is transferred into yeast competent cells. pGBKT7 empty vector was used as a control while transforming yeast strains. PPLS1 transcriptional activation activity was tested on the corresponding deficient medium. The results show that 8 yeasts transformed with the fusion expression vector and the pGBKT7 empty vector can grow normally on the SD/-Trp plate; PPLS1 containing 202-377aa region on SD/-Trp + X- α -gal/Aba plates^1-571、PPLS1^202-377、PPLS1^1-377And PPLS1^202-429The fusion expressed protein was able to activate the expression of AUR1-C and MEL1 reporter genes in yeast, which normally grew blue (FIG. 11). The results indicate that PPLS1 has transcriptional activation activity, and that 202-377aa is located in the WD/AD domain, which is essential for PPLS1 transcriptional activation.

Next, 5 different SiMYB85 domains (full-length SiMYB85 and a partially truncated SiMYB85 fragment, in turn designated as SiMYB85^1-268、SiMYB85^1-64、SiMYB85^65-118、SiMYB85^119-268And SiMYB85^1-119FIG. 12) are respectively connected with GAL4-DNA binding structural domain in pGBKT7 vector to construct yeast fusion expression vector, and then the yeast fusion expression vector is transferred into yeast competent cells. pGBKT7 empty vector was used as a control while transforming yeast strains. In pairSimYB85 transcriptional activation activity was tested on the corresponding deficient medium. The results show that 5 yeasts transformed with the fusion expression vector and the pGBKT7 empty vector can grow normally on the SD/-Trp plate; on SD/-Trp + X- α -gal/Aba plates, only SiMYB85^119-268The fusion expressed protein was able to activate the expression of AUR1-C and MEL1 reporter genes in yeast, which grew normally and turned blue (FIG. 13). The results indicated that the SiMYB85 protein had no transcriptional activation activity, but the C-terminal domain of 119-267aa had transcriptional activation activity.

Example 7: tobacco transient expression experiments prove that two genes of the ppls1 and the Simyb85 are expressed together to play a role

To demonstrate the function of both genes Ppls1 and Simyb85 in anthocyanin biosynthesis, we injected Agrobacterium GV3101 carrying Ppls1, Simyb85, Ppls1+ Simyb85 and the empty vector, respectively, into nicotiana benthamiana leaves. The results show that neither gene Ppls1 nor Simyb85 injected alone can affect the color of the nicotiana benthamiana leaves, similar to the empty vector injection phenotype; co-expression of both Ppls1 and Simyb85 genes affected the tobacco leaves to purple on day 5 post injection. The anthocyanin content in the tobacco leaves was measured (fig. 14, 1 indicates an empty vector, 2 indicates Ppls1, 3 indicates Simyb85, and 4 indicates Ppls11+ Simyb85), and the anthocyanin content in the leaves co-expressed with Ppls1 and Simyb85 was the highest, 2 times that of the leaves expressing only the Simyb85 gene, and 4 times that of the leaves expressing only the Ppls1 gene.

Example 8: two proteins, PPLS1 and SiMYB85, interact with each other

To determine whether the two proteins PPLS1 and SiMYB85 interacted in vivo, we performed a yeast two-hybrid. Ligation of the full-length CDS sequence of SiMYB85 into the pGBKT7(BD) vector to construct BD-SiMYB85^1-268Recombinant plasmid, PPLS1 full-length CDS sequence connected to pGADT7(AD) vector to construct AD-PPLS1^1-571Recombinant plasmid, BD-SiMYB85^1-268And AD-PPLS1^1-571The two recombinant plasmids were co-transformed into the yeast strain Y2HGold, BD-SiMYB85^1-268+AD、BD+AD-PPLS1^1-571BD + AD vector as negative control. As shown in FIG. 15, the transformants were able to grow on the defective medium SD/-Leu/-Trp/-His/-ANormal growth on de (SD/-LTHA), but transformants with neither single gene nor empty vector, showed that PPLS1 was able to interact with both proteins of SiMYB 85.

Example 9: application of genes Ppls1 and Simyb85 for controlling leaf sheath and leaf pillow color of Shilixiang in identifying true and false hybridization and millet hybridization

A. Sowing male parent Shilixiang (purple leaf sheath leaf pillow) and female parent Yugu No. 1 (green leaf sheath leaf pillow) at intervals of one-row Shilixiang and two-row Yugu No. 1, wherein the sowing time interval of the two-row Yugu No. 1 is 5-7 days;

B. carrying out male-female hybridization according to a conventional hybridization method of the millet, namely: the ear of Yugu No. 1 of the female parent with the total number of opened florets accounting for 10-15% of the total number of spikelets is treated as follows: removing partial ear codes of the female parent ears, reserving 15-25 ear codes of each female parent ear, and removing all open florets and underdeveloped flowers in the reserved ear codes.

C. Castration treatment is carried out on the female parent reserved spike codes: soaking seeds in 42 deg.C hot water for 8 min;

D. 2-3 male parent florescence-meeting shili spike and the treated female parent spike are sleeved together by a sulfuric acid paper bag, and the bag is knocked to pollinate for 7 days between 9 and 10 points in the morning every day;

E. harvesting seeds of the current hybrid generation F0 on the female parent plant;

F. seeds of F0 generation are planted, the plant is F1 generation, the F1 generation of purple sheath leaf pillow is the filial generation of true hybrid, the F1 generation of green sheath leaf pillow is the filial generation of false hybrid, and the filial generation is discarded.

The gene for controlling the color of the leaf sheath and the leaf occipital of the millet is the main reason for determining that the color of the leaf sheath and the leaf occipital of the millet is purple in the growth process, can be used as a good breeding indicator in breeding, can visually judge whether hybridization succeeds or not, and can identify true and false hybrid varieties. Through long-term research and analysis, the SNP sites of the PPLS1 and SiMYB85 sequences are the genes as long as the amino acid sequence is not influenced, the same action and effect as those of the genes can be realized, and the genes belong to the protection scope of the patent.

SEQUENCE LISTING

<110> millet institute of academy of agriculture, forestry and science of Hebei province

<120> gene for controlling color of sheath and occipital color of millet and application thereof in breeding

<160> 2

<210> 1

<211> 1716

<212> DNA

<400> 1

atggcactac cagcttcccc agttcaggaa gaactgcagc cggagagtca attgaggaac 60

catcttgctg cagccgtgag gagcatcaac tggacttatg ccctcttctg gtccatttca 120

agcactcagc caggattcct aacgtggacg gatgggttct acaacggcga ggtgaagacg 180

aggaagatcg ccaactctgc ggagctgacc gccgaccagc tcgtcatgca gcgaagcgaa 240

cagctgcggg agctctacga ggccctcctc tccggcgagt gcgaccgccg gacggcgcgg 300

ccggtcgcct cgctgtcgcc ggaggacctc ggcgacaccg agtggtacta cgtggtctgc 360

atgacctacg ccttccggcc aggccaaggg ttgccgggaa ggagctttgc tagcaacgaa 420

cgtgtctgga tgcggaactc tcacctcgcc gacagcaaat ccttcccccg cgcgctcctg 480

gcgaagagcg cgtccattca gacaatcgtc tgcatcccgt tcatgagcgg cgtgcttgag 540

ctggggacaa ctgatgcggt ggtggaggat ccaagcttgg tcagccgagc cactgcatct 600

ttctgggaga tgcagtttcc ggcatgctcg caggagccga gctccagccc gtcagcaaac 660

gaaaccggca agcccgccga cattatcgtg ttcgaggacc tcgatcacat tgccatggaa 720

gcgatgatcg ccggcggaca ggagctaggc gaagcggaga gcctgtcaga tggaaccctc 780

gagcaaatca ccaaggagat cgacgaattc tacagcctct gcgaggaaat ggacgtgcag 840

ccactcgagg atacctggat catggacggg tctttcgaag tcccctcttc gccgcagccg 900

gcgccggggc ctgctactac taacgccgcc gccacctcaa gcgctctcgt tgacggctct 960

cgcgcgacga gctttacggc ctgggcgagg ccggagtcgg actccgatga agtggctgtg 1020

ccggtcgtcg aagagccaca gaagctgctg aagaaagcgg tggccggcgg agcttgggcg 1080

gcgaacaatg gcggcggcgg cacgacgaga atggcccaag aaagtggcgt caagaaccac 1140

gtcatgtcgg agaggaagcg ccgggaaaag ctcaacgaga tgttcctcgt tctcaagtca 1200

ttggttccct ccattcacaa ggtggacaaa gcatcgatcc ttgccgaaac gatagcgtac 1260

ctcaaggagc ttcaacgaag ggtacaagag ctagaatcca gcagggaacc cataatctcg 1320

cgcccatccg aaacaacgag ggcaacaagg cgccatgacg atgaggccgt caggaagaaa 1380

gtctgtgccg cgggctccaa gaggaagggc tcggagctcg gcggtgacgt ggagagggag 1440

cacccacggg ccctctccat ggacggcacc agcaacgtca ccgtcaccgt ctcggacaag 1500

gaggtgctcg tggaggtgca gtgccggtgg gaagagcttc tgatgacgcg agtgttcgac 1560

gcaatcaaga gcctgcaact ggacgttctc tcggttcagg catcggcacc ggatgggttc 1620

atggggctga agatacgggc tcagtttgcc ggctcagctg ccgttgtgcc gtggatgatc 1680

agcgaggccc tccgtaaagc tatagggaag cgatga 1716

<210> 2

<211> 807

<212> DNA

<400> 2

atggggagga gggcgtgctg tgccaaggaa gggttgaaga gaggggcgtg gacgagcaag 60

gaagacgaca tcctggccgc ctacgtcaag gcccatggcg aaggcaaatg gagggaggtc 120

ccccagaaag ccggtttgcg ccggtgcggc aagagctgcc ggctgcggtg gctcaactac 180

ctccggccga acatcaagcg cggcaacatc tccgacgagg aggaggacct catcatccgg 240

ctccacaagc tcctcggcaa caggtggtcg ctgatcgcag ccaggctgcc gggccgaaca 300

gacaatgaaa tcaagaacta ctggaacagc acgctcggcc ggagggccgg cgccggcgga 360

ggcggcagca gggtcgtcgt cttcgggaca cccgacaccg gctcacactc acaagccaca 420

ccggcggcgt cgggcagctg cgagaacggc gccgccgctc atcgcacgga ccccgattca 480

gctggctccg ccgccgggac ggcctcggca gcggcggccg tgtgggcgcc caaggccgtg 540

cggtgcacgg gccgcctctt cttccaccgg gacctgctgg aggcgccgcc cgcgagcgag 600

acgccgacgg ccgggggaga agtagggggg tcgggagaag ggtcgtcgga cgactgcagc 660

tcggcggcgt cgaccttcgc gggcgtcatc gacgagccgt gcttctccgg cggcggctgg 720

atggacgacg tgagggccct ggcgtcgttc ctcgagtccg acgaggaatg ggtcgtccgg 780

tgtcagatgt ccgagcagcc ggcttag 807

Claims

1. The application of the gene combination for controlling the color of the leaf sheath and the leaf pillow of the millet in the hybridization breeding of the millet is characterized in that: the gene combination comprises a Ppls1 gene and a Simyb85 gene, wherein the coding sequence of the Ppls1 gene is shown as a sequence table SEQ ID NO: 1, the coding sequence of the Simyb85 gene is shown in a sequence table SEQ ID NO: 2, respectively.