CN116041466A - Barley grain black character HvBlp gene and related molecular marker and application thereof - Google Patents
Barley grain black character HvBlp gene and related molecular marker and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology and genetic breeding, and discloses black character of barley seedsHvBlpGene, related molecular marker and application thereof, and barleyHvBlpThe nucleotide sequence of the gene is shown as SEQ ID NO.1, and the encoding protein is shown as SEQ ID NO. 2. The gene is prepared in black variety barley by using BSMV-VIGS technology in early stage of groutingSilencing in ears, and finding that the color of seeds in all silencing treated ears becomes light, shows that the gene is involved in regulating and controlling the biosynthesis of melanin in barley grains. Therefore, the invention has good application prospect in cultivating new black barley varieties. In addition, based on the gene CDS and the gene upstream sequence, the invention also provides three pairs of molecular markers as identification combinations, which can be used for carrying out initial screening on breeding materials, achieves the aim of molecular marker assisted breeding, accelerates the breeding process and has important theoretical and practical significance.
Description
Technical Field
The invention relates to the technical fields of molecular biology and genetic breeding, in particular to a barley grain black character HvBlp gene and related molecular markers and application thereof.
Background
Melanin is a complex polymeric pigment that is widely found in nature, including humans, animals, plants and microorganisms. Plant melanin has been shown to have biological activities such as antioxidant, antibacterial, anticancer, anti-inflammatory, immunomodulating and anti-aging. For example, black tea melanin has liver protecting effect (Sava et al, 2003). Sesame melanin can be used in clinical cancer treatment (Chu et a.l., 2016). Studies by Al-obes et Al show that nigella sativa seed melanin can inhibit colorectal cancer cell proliferation by altering redox balance and modulating MAP K signaling pathways (Al-obes et Al 2020). Studies by El-Obeid et al indicate that black grass seed melanin is capable of modulating the expression of cell growth factors, has immunomodulatory activity, and may be helpful in future cytokine production imbalance and cancer-related disease treatment (El-Obeid et al, 2006). In addition, melanin obtained from buckwheat hulls has been successfully used as a food supplement to improve food nutritional value and antioxidant activity (kortacheva et al 2021).
Barley is used as an important medical and edible coarse cereal crop, and the grains are rich in dietary fibers, beta-glucan and other bioactive components. Black barley is formed due to melanin accumulation in the barley seed coats and hulls (Shoeva et al 2020). The black barley not only has various advantages of common barley, but also contains more abundant polyphenols, has stronger scavenging ability of superoxide radical, hydroxyl radical and 2, 2-diphenyl-1-picrylhydrazine radical, iron reduction antioxidation ability and moderate metal ion chelating ability, and can be used as a natural antioxidant. Therefore, understanding the mechanism of melanin formation in barley kernels and increasing the content of melanin in barley kernels are of great significance in improving the nutritional value of grains and guaranteeing the nutritional health of the national people.
In previous studies, it was reported that the black grain shape of barley was controlled by a gene named HvBlp, which was located in a physical region of 0.807Mb on chromosome 1 of barley, and at least 21 candidate genes were predicted in this region. However, because the segment is longer, the difference among different varieties of the segment sequences is smaller, the segment is closely linked, the probability of recombination exchange is smaller, the difficulty of further positioning work is high, and Blp has not been cloned and reported until the application date.
In the invention, the applicant clones the barley Hv Blp gene by using a map-based cloning method by further expanding the number of genetic groups, and verifies the function of regulating and controlling the black grain traits of the barley by using a BSMV-VIGS silencing technology. Provides related genes and theoretical basis for cultivating new varieties of black barley. In addition, by utilizing the CDS sequence of the HvBlp gene and the gene upstream fragment, a molecular marker closely linked with the black kernel shape of the barley is developed, and can be used for screening the early generation of black barley varieties, and the breeding efficiency can be improved.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to provide a gene HvBlp which is cloned from black barley and can regulate and control the shape of black barley kernels, and provides related genes for cultivating black healthy barley, wherein the protein coded by the gene is shown as SEQ ID NO. 2.
It is another object of the present invention to provide an InDel dominant molecular marker primer combination designed for the resulting black barley gene, for identification of black barley grain characteristics.
The final object of the invention is to provide the application of the HvBlp gene or related identification primer thereof in the color breeding of barley grains.
In order to achieve the above object, the present invention adopts the following technical measures:
the applicant constructs genetic groups through black kernel barley W1 and yellow kernel barley Hindmarsh, and locates barley HvBlp gene by using a map cloning method, wherein the protein encoded by the gene is shown as SEQ ID NO.2, and the preferable gene is shown as SEQ ID NO. 1.
The application of the gene for encoding the protein shown in SEQ ID NO.2 or the protein shown in SEQ ID NO.2 in the breeding of barley seed color also belongs to the protection scope of the invention;
the color of the seeds is black or yellow.
The application is that the gene of the black kernel barley for encoding the protein shown in SEQ ID NO.2 is silenced, mutated or interfered to lose the original function, so as to obtain the transgenic barley with light color;
for the above gene silencing, the protection scope of the present invention also includes a gamma vector comprising the gene shown in SEQ ID NO. 3.
The protection scope of the invention also comprises:
aiming at the difference of black grain barley and yellow grain barley HvBlp genes, the designed InDel dominant molecular marker primer combination is:
forward primer BL 1-F5'-GCCATGCATGAAGATCGAGC-3'
Reverse primer BL 1-F5'-GGAGCGCTGGTTGATGATCT-3'
Forward primer BL 2-F5'-TCCTCCCTCCATCACTCC-3'
Reverse primer BL 2-F5'-ATGCGCTCATGCACCTCT-3'
Forward primer BL 3-F5'-GTATCTACCCTCAACTTCGGATCC-3'
Reverse primer BL 3-F5'-TCAGAAGTCATGAATAATCTTCT GC-3'.
The amplification length of the BL1 InDel molecular marker is 146bp and/or 139bp, if the amplification gene fragment length is 146bp, the single plant is homozygous for the yellow barley seed, if the amplification gene fragment length is 139bp, the single plant is homozygous for the black barley seed, and if the amplification gene fragment length is 146bp and 139bp, the single plant is heterozygous for the black barley seed.
The BL2 InDel molecular marker has an amplification length of 1393bp, if the amplification gene fragment has a length of 1393bp, the black barley seed single plant is homozygous or heterozygous, and if the amplification gene fragment has a deletion, the black barley seed single plant is homozygous.
The BL3 InDel molecular marker is homozygous or heterozygous black barley grain single plant if the length of the amplified gene fragment is 491bp, and is homozygous yellow barley grain single plant if the amplified gene fragment is missing.
The invention also provides application of the primer combination in the color phenotype of barley kernels.
Compared with the prior art, the invention has the following advantages:
the invention discloses a barley HvBlp gene for the first time, and verifies the application of the barley HvBlp gene in regulating and controlling the formation of black grains. The result of VIGS silencing technology proves that the silencing HvBlp gene can obviously reduce the color of barley grains. Therefore, the invention provides a new variety of black kernel barley which can be cultivated by utilizing the HvBlp gene. In addition, the invention also provides a group of InDel molecular markers related to the black character of the barley seeds and application thereof in the breeding of the black barley seeds. Effectively solves the problem of early genotype identification in the black kernel barley breeding process.
Drawings
Fig. 1 is a schematic diagram of the yellow kernel barley female parent Hindmarsh and the barley male parent W1.
FIG. 2 shows the HvBlp gene transmembrane domain (A) and the functional domain prediction map (B).
FIG. 3 shows a graph of black barley phenotype change after VIGS silencing.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. In addition, the technical scheme of the invention is a conventional scheme in the field unless specifically stated; the reagents or materials, unless otherwise specified, are commercially available.
Example 1: cloning of barley HvBlp Gene coding region
A suitable amount of black kernel barley W1 (Long et al, 2018) seed was taken into a planter, and placed in a greenhouse for growth. And (5) in the grouting period (25 days after flowers), peeling the seed coats of the black seeds, extracting RNA from the seed coats and transferring to obtain cDNA. Then, PCR amplification is carried out on the HvBlp gene as a template, and the HvBlp forward primer: 5'-ATGAGCGCATCGCCGGACGTGCG-3', reverse primer: 5'-TCAGAAGTCATGAATAATCTTCTGC-3', the nucleotide sequence shown in SEQ ID NO.1 and encoding the protein shown in SEQ ID NO.2 are finally obtained by sequencing. The HvBlp protein sequence was predicted and analyzed by SMART (htt p:// SMART. Emmbl-heidelberg. De /) and InterPro (http:// www.ebi.ac.uk/Interpro /) databases, and the results showed that the protein belongs to 9 transmembrane structures (FIG. 2A) and AA_trans protein domains (FIG. 2B) of the transmembrane protein.
Example 2: BSMV-VIGS method for verifying HvBlp gene function
The VIGS silencing primer was designed based on the CDS sequence: hvBlp-VIGS-F TTTTTTTTTTTTTAGATACTGG AGGGGGACAA; hvBlp-VIGS-R ATTCTTCTTCCGTTGGCCGTAGCAGAAAAGGT. And (3) carrying out PCR amplification by using the W1 seed coat cDNA as a template to obtain a 310bp silent gene fragment (SEQ ID NO. 3), and finally reversely inserting the silent gene fragment into an RNA gamma vector (He et al 2015) through homologous recombination to obtain an RNA gamma HvBlp recombinant vector and carrying out sequencing verification. And (3) single enzyme digestion of RNA alpha, RNA gamma and RNA gamma in the VIGS system is carried out by utilizing MluI restriction enzyme, single enzyme digestion is carried out on beta carrier by utilizing SpeI restriction enzyme, and after enzyme digestion is finished, the linearized carrier is recovered and the product is purified. The purified RNA alpha, RNA beta, RNA gamma and RNA gamma HvBlp linearized products were transcribed in vitro using the RiboMAXTM LargeScale RNA Production System-T7kit and the Ribo m7G Cap Analo g kit kit (Promega, USA). Finally, the W1 green spike (10 days after flowers) is infected according to the BSMV-VIGS routine technical operation manual, and the infection is carried out twice in succession at intervals of 7 days, and the result shows that the color of the seed with RNA gamma: hvBlp infection silence is obviously lighter than that of a control (RNA gamma blank vector infection) (figure 3).
Example 3: development and application of InDel molecular marker for identifying black kernel characteristics of barley
3.1 design of a set of InDel molecular marker primers for identification of the black kernel shape of barley:
comprises three pairs of primers: BL1, BL2 and BL3 correspond specifically to the following three pairs:
forward primer BL 1-F5'-GCCATGCATGAAGATCGAGC-3'
Reverse primer BL 1-F5'-GGAGCGCTGGTTGATGATCT-3'
Forward primer BL 2-F5'-TCCTCCCTCCATCACTCC-3'
Reverse primer BL 2-F5'-ATGCGCTCATGCACCTCT-3'
Forward primer BL 3-F5'-GTATCTACCCTCAACTTCGGATCC-3'
Reverse primer BL 3-F5'-TCAGAAGTCATGAATAATCTTCT GC-3'
The amplification length of the BL1 InDel molecular marker is 146bp and/or 139bp, if the amplification gene fragment length is 146bp, the single plant is homozygous for the yellow barley seed, if the amplification gene fragment length is 139bp, the single plant is homozygous for the black barley seed, and if the amplification gene fragment length is 146bp and 139bp, the single plant is heterozygous for the black barley seed.
The BL2 InDel molecular marker has an amplification length of 1393bp, if the amplification gene fragment has a length of 1393bp, the black barley seed single plant is homozygous or heterozygous, and if the amplification gene fragment has a deletion, the black barley seed single plant is homozygous.
The BL3 InDel molecular marker is homozygous or heterozygous black barley grain single plant if the length of the amplified gene fragment is 491bp, and is homozygous yellow barley grain single plant if the amplified gene fragment is missing.
3.2 Co-segregation verification of recombinant inbred populations and InDel molecular markers
(1) Hybridization is carried out by taking yellow kernel barley Hindmarsh as female parent and black kernel barley W1 as male parent to obtain F1 filial generation, and backcross is carried out with Hindmarsh for two times to obtain BC 2 F 1 Finally, obtaining BC through multiple selfing 2 F 3 A population.
(2) Material planting and phenotype identification
2.1 the parents Hindmarsh and W1, and 50 black and yellow offspring from the constructed colony materials are randomly sown in plug trays, placed in a greenhouse for growth, and subjected to conventional cultivation management until maturity.
2.2 fresh tender leaves are taken in the seedling stage, and sample DNA is extracted by adopting a CTAB method.
2.3 after barley grain ripening, the grain color was observed and recorded by human eyes.
(3) PCR detection
Detection was by ordinary PCR amplification and agarose gel electrophoresis. Genotype of barley grain color trait was judged based on banding of PCR products, as shown in Table 1, 100 BC randomly extracted by the marker pairs of the present invention 2 F 3 The coincidence rate of the phenotype and the genotype of the single plant of the sample is 100 percent.
TABLE 1 population individual identification results
Note that: w represents the genotype of the black kernel parent W1, H is the genotype of the yellow kernel parent Hindmarsh, and WH is the heterozygous genotype.
Claims (8)
1. An isolated protein for controlling black character of barley grains, which is shown in SEQ ID NO. 2.
2. A gene encoding the protein shown in SEQ ID NO. 2.
3. The gene according to claim 2, wherein said gene is shown in SEQ ID NO. 1.
4. Use of the protein of claim 1 or the gene of claim 2 in barley grain color breeding.
5. The use according to claim 4, wherein the method of breeding barley with lighter grain color is to silence, mutate or interfere with the gene encoding the protein shown in SEQ ID NO.2 in black grain barley to lose its original function.
6. The use according to claim 5, in a gene silencing mode, comprising the use of a gamma vector comprising the gene of claim SEQ ID NO. 3.
7. InDel dominant molecular marker primer combinations for identifying barley grain color:
forward primer BL 1-F5'-GCCATGCATGAAGATCGAGC-3'
Reverse primer BL 1-F5'-GGAGCGCTGGTTGATGATCT-3'
Forward primer BL 2-F5'-TCCTCCCTCCATCACTCC-3'
Reverse primer BL 2-F5'-ATGCGCTCATGCACCTCT-3'
Forward primer BL 3-F5'-GTATCTACCCTCAACTTCGGATCC-3'
Reverse primer BL 3-F5'-TCAGAAGTCATGAATAATCTTCT GC-3'.
8. Use of the primer combination of claim 7 for identifying a barley grain color phenotype.
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| JP2005224221A (en) * | 2004-02-16 | 2005-08-25 | Japan Science & Technology Agency | Genetic marker linked with locus ruling lemma color and utilization of the same |
| CN106811464A (en) * | 2015-12-01 | 2017-06-09 | 深圳华大农业与循环经济科技有限公司 | With the molecular labeling SVmc1 of hasked millet color gene close linkage |
| WO2018113702A1 (en) * | 2016-12-21 | 2018-06-28 | Institute Of Crop Sciences, The Chinese Academy Of Agricultural Sciences | Plant grain trait-related protein, gene, promoter and snps and haplotypes |
| CN114015701A (en) * | 2021-11-23 | 2022-02-08 | 四川农业大学 | Molecular marker for detecting barley grain shrinkage character and application thereof |
| CN114836450A (en) * | 2022-06-30 | 2022-08-02 | 中国农业科学院作物科学研究所 | Colored barley grain anthocyanin transport related gene HvGST and application thereof |
| CN115369181A (en) * | 2022-02-16 | 2022-11-22 | 湖北省农业科学院粮食作物研究所 | SNP (Single nucleotide polymorphism) marker related to barley ear and grain morphology, primer set and application thereof |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005224221A (en) * | 2004-02-16 | 2005-08-25 | Japan Science & Technology Agency | Genetic marker linked with locus ruling lemma color and utilization of the same |
| CN106811464A (en) * | 2015-12-01 | 2017-06-09 | 深圳华大农业与循环经济科技有限公司 | With the molecular labeling SVmc1 of hasked millet color gene close linkage |
| WO2018113702A1 (en) * | 2016-12-21 | 2018-06-28 | Institute Of Crop Sciences, The Chinese Academy Of Agricultural Sciences | Plant grain trait-related protein, gene, promoter and snps and haplotypes |
| CN114015701A (en) * | 2021-11-23 | 2022-02-08 | 四川农业大学 | Molecular marker for detecting barley grain shrinkage character and application thereof |
| CN115369181A (en) * | 2022-02-16 | 2022-11-22 | 湖北省农业科学院粮食作物研究所 | SNP (Single nucleotide polymorphism) marker related to barley ear and grain morphology, primer set and application thereof |
| CN114836450A (en) * | 2022-06-30 | 2022-08-02 | 中国农业科学院作物科学研究所 | Colored barley grain anthocyanin transport related gene HvGST and application thereof |
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