CN105132440B - With the wheat leaf blade chlorophyll content and relevant gene of grain weight, its Indel labels and application - Google Patents

Abstract

The invention relates to a gene TaBAS1‑2B related to wheat leaf chlorophyll content and grain weight and its Indel marker. The present invention isolates and clones the gene TaBAS1‑2B related to the chlorophyll content of wheat leaves and grain weight from wheat for the first time, and according to the sequence of the cloned TaBAS1‑2B gene, two varieties of 'Jing 411' and 'Hongmangchun 21' Developed a polymorphic Indel marker during the period, and analyzed its effect on the phenotype in 194 wheat natural varieties, and finally developed an Indel marker that co-segregated with the gene TaBAS1‑2B and was closely related to wheat leaf chlorophyll content and grain weight function flags. The Indel marker can explain 8.2% and 4.4% of the phenotypic variation of flag leaf chlorophyll content and thousand-grain weight after anthesis in wheat, respectively, and the band with a molecular weight of 494bp (Seq ID No.3) has a synergistic effect on the increase of grain weight , the band with a molecular weight of 493bp (Seq ID No.4) has a synergistic effect on the reduction of grain weight.

Description

Genes related to wheat leaf chlorophyll content and grain weight, their Indel markers and application

technical field

The invention relates to the fields of genetic engineering and molecular biology, in particular to a gene related to wheat leaf chlorophyll content and grain weight, its Indel marker and application.

Background technique

Wheat is one of the most important food crops in the world. With the increase of population, the reduction of arable land and the continuous increase of food production costs, high-yield and ultra-high-yield breeding has become an urgent problem to be solved in my country's wheat breeding. The growth process of wheat is a process of continuous material production through photosynthesis, and green leaves are the main source of material production. Under suitable conditions, approximately 70-90% of wheat grain yield during grain filling is derived from photosynthetic products produced by flag leaves after anthesis (Austin et al., 1997; Bidinger et al., 1997). Therefore, prolonging the functional period of wheat flag leaves, promoting leaves to produce more photosynthetic products, and thus increasing yields are the basis for high yields of wheat.

In higher plants, chlorophyll is an important photosynthetic pigment in chloroplasts, which plays an important role in the absorption and use of light energy. Studies have shown that the decline of chlorophyll content in leaves during the filling period significantly affects the photosynthetic rate of plants, which directly leads to the decline of photosynthetic efficiency (Araus et al., 1997). Wang et al. (2008) pointed out in their research that increasing chlorophyll content is an effective way to increase plant biomass and grain weight.

After being subjected to biotic and abiotic stress, the reactive oxygen species (reactive oxygen species, ROS) content in the plant will increase, which will cause poisoning to cells and lead to chloroplast damage, resulting in a decrease in photosynthetic rate, which will eventually affect grain weight and yield. Plants have formed a relatively complete ROS scavenging enzyme system during the evolution process, among which peroxiredoxin (Prx) plays an important role in protecting chloroplasts and other organelles from oxidative damage. Plant peroxiredoxin BAS1 is a chloroplast protein encoded by a nuclear gene and belongs to the AhpC family. The protein is a sulfhydryl-dependent peroxidase that catalyzes the reduction of hydrogen peroxide via Cys residues, and an NADPH-dependent chloroplast thioredoxin reductase that maintains the reduced state of BAS1. Ruiz et al. (2006) confirmed in rice that the NTRC-BAS1 pathway is an efficient redox system for chloroplast protection from oxidative damage.

Although there are many studies on the structure and function of peroxiredoxin in plants, its specific mechanism of action in plants is still not completely clear. Research reports on Prx in wheat are relatively rare, and no relevant research reports on wheat BAS1 gene have been found so far. Whether the expression of BAS1 gene affects the content of chlorophyll in leaves and whether it is related to grain weight is still unclear. Therefore, the study of peroxidoreductase gene BAS1 provides a basis for further understanding the mechanism of ROS scavenging enzyme system, and it is also of great significance for studying its role in improving wheat yield and grain weight.

Contents of the invention

The purpose of the present invention is to provide a gene TaBAS1-2B related to wheat leaf chlorophyll content and grain weight.

Another object of the present invention is to provide an Indel marker co-segregated with the wheat gene TaBAS1-2B and its application.

In order to realize the purpose of the present invention, the gene TaBAS1-2B related to the chlorophyll content and grain weight of wheat leaves provided by the present invention is obtained through electronic cloning combined with conventional PCR methods. According to the CDS sequence (GenBank: AB000405.1) of wheat thioredoxin peroxidase (Thiol-specific antioxidant protein) published on NCBI, the wheat EST database was searched, and the screened EST sequences (CA484935.1, CK214438.1 , CK217637.1) for electronic assembly, using the spliced sequence as a template, using Primer Premier 5.0 software to design three pairs of primers B1, B2 and B3 for the acquisition of wheat BAS1 gene cDNA, and the other two pairs of primers G1 and G2 for wheat BAS1 gene (see Table 1 for primer information).

Table 1 Primers used to amplify wheat TaBAS1-2B gene and its cDNA sequence

The PCR reaction uses the cDNA and genomic DNA of the wheat variety 'Jing 411' as templates, and then the products amplified by primers B1, B2 and B3 are sequentially digested and ligated, and finally the cDNA sequence of the wheat BAS1 gene is obtained, with a size of 846bp , the products amplified by primers G1 and G2 were sequentially digested and ligated to finally obtain the complete sequence of the wheat BAS1 gene with a size of 2856bp. The gene was located on the wheat 'China Spring' lacking tetrasomy material, and the gene was located on the wheat chromosome 2B, and the gene was named TaBAS1-2B.

Specifically, the nucleotide sequence of the gene TaBAS1-2B related to wheat leaf chlorophyll content and grain weight provided by the present invention is:

i) the nucleotide sequence shown in Seq ID No.1; or

ii) The nucleotide sequence shown in Seq ID No.1 is substituted, deleted and/or increased by one or more nucleotides and expresses the same functional protein nucleotide sequence; or

iii) a nucleotide sequence that hybridizes to the sequence shown in Seq ID No.1 under stringent conditions;

The stringent condition is hybridization at 65° C. in 0.1×SSPE containing 0.1% SDS or 0.1×SSC solution containing 0.1% SDS, and the membrane is washed with this solution.

The cDNA sequence of the gene TaBAS1-2B is shown in Seq ID No.2.

The invention also provides vectors, engineering bacteria and transgenic cell lines containing the gene TaBAS1-2B.

According to the cloned gene TaBAS1-2B sequence, a polymorphic Indel marker was developed between the two varieties of 'Jing 411' and 'Hongmangchun 21', and a pair of Indel markers were designed using Primer Premier 5.0 software to amplify all The primers for the above-mentioned Indel markers were analyzed in 194 natural varieties of wheat to analyze their effects on phenotypes, and finally a functional marker co-segregated with the gene TaBAS1-2B and closely related to wheat leaf chlorophyll content and grain weight was developed .

The pair of primers used to amplify the Indel marker is:

Upstream primer F: 5'-CGCAGTGCCTGTCGTTTC-3'(Seq ID No.5)

Downstream primer R: 5'-TCACATACTTCTTTCCCAAT-3'(Seq ID No.6)

The Indel marker characteristic band associated with the higher chlorophyll content and higher grain weight of wheat leaves amplified by the primer pair is 494bp, and its nucleotide sequence is as shown in Seq ID No.3; The characteristic band of Indel marker associated with lower chlorophyll content and lower grain weight in wheat leaves is 493bp, and its nucleotide sequence is shown in Seq ID No.4.

The specific technical route of the gene TaBAS1-2B clone and its Indel marker development of the present invention is shown in FIG. 1 .

The invention also provides the application of the Indel marker in screening wheat germplasm resources with leaves having higher chlorophyll content and higher grain weight. Including the following steps:

1) extracting the genomic DNA of the plant to be tested;

2) using the genomic DNA of the plant to be tested as a template, using the primers F and R (Seq ID No.5 and 6) to perform a PCR amplification reaction;

3) Detecting the PCR amplification product, if the characteristic band of the nucleotide sequence shown in Seq ID No.3 can be amplified, the plant to be tested is a wheat resource with higher chlorophyll content in leaves and higher grain weight.

Among them, the amplification system used in the PCR reaction is calculated as 10 μl: template DNA 100ng, 10μM primers F and R 0.2μl, 2.5mM dNTP 0.8μl, 5U/μl Taq DNA polymerase ^0.12μl , 10 ×PCR reaction buffer 1.0 μl, make up to 10 μl with ddH ₂ O.

The amplification program of the PCR reaction was: 94°C for 5 minutes; 94°C for 50 seconds, 58°C for 50 seconds, 72°C for 3 minutes, 36 cycles; 72°C for 10 minutes.

Preferably, in step 3), the PCR amplification product is detected by 6% denaturing polyacrylamide gel electrophoresis, and then developed by silver staining.

The present invention further provides the application of the Indel marker in wheat molecular marker assisted breeding.

The present invention isolates and clones the gene TaBAS1-2B related to wheat leaf chlorophyll content and grain weight from wheat for the first time. Developed a polymorphic Indel marker, and analyzed its effect on phenotype in 194 wheat natural varieties, and finally developed an Indel marker that co-segregated with the gene TaBAS1-2B and was closely related to wheat leaf chlorophyll content and grain weight function flags. The Indel marker can explain 8.2% and 4.4% of the phenotypic variation of flag leaf chlorophyll content and thousand-grain weight after anthesis in wheat, respectively, and the band with a molecular weight of 494bp (Seq ID No.3) has a synergistic effect on the increase of grain weight , the band with a molecular weight of 493bp (Seq ID No.4) has a synergistic effect on the reduction of grain weight. The development of this molecular marker provides a feasible way for high-yield wheat molecular assisted breeding.

Description of drawings

Fig. 1 is a specific technical roadmap for the development of the gene TaBAS1-2B clone and its Indel marker of the present invention.

Figure 2 is the allelic type analysis of Indel markers in different wheat varieties in Example 3 of the present invention; wherein, lane 1 is Henong 972, lane 2 is Zhoumai 16, lane 3 is Baomai 8, and lane 4 is Womai No. 8, lane 5 is Annong 9267, lane 6 is Lumai 23, lane 7 is Jinmai 31, lane 8 is Zhoumai 18, lane 9 is Annong 1014, lane 10 is Aikang 58, and lane 11 is Chuanmai 42. Lane 12 is Jimai 20, Lane 13 is Panmai 5, and M is DNA Marker.

Fig. 3 is the result of positioning the gene TaBAS1-2B using wheat 'China Spring' missing tetrasome material in Example 3 of the present invention; wherein, lane 1 is wheat 'China Spring' (CS), and lanes 2-7 are respectively CS N2AT2B, CS N2AT2D, CSN2BT2A, CS N2BT2D, CS N2DT2A, CS N2DT2B.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular cloning experiment manual (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions.

The sequencing in the following examples was completed by Sangon Bioengineering Shanghai (Co., Ltd.).

The acquisition of embodiment 1 wheat TaBAS1-2B gene

The gene TaBAS1-2B related to wheat leaf chlorophyll content and grain weight was obtained by electronic cloning combined with conventional PCR. According to the CDS sequence (GenBank: AB000405.1) of wheat thioredoxin peroxidase (Thiol-specificantioxidant protein) published on NCBI, the wheat EST database was searched, and the screened EST sequences (CA484935.1, CK214438.1, CK217637.1) for electronic splicing, using the spliced sequence as a template, using Primer Premier 5.0 software to design three pairs of primers B1, B2 and B3 for the acquisition of wheat BAS1 gene cDNA, and the other two pairs of primers G1 and G2 for wheat BAS1 gene cDNA See Table 1 for primer information.

Table 1 Primers used to amplify wheat TaBAS1-2B gene and its cDNA sequence

The PCR reaction was carried out on the BIO-RAD MyCycler 1.0 PCR instrument using the cDNA and genomic DNA of the wheat variety 'Jing 411' as templates. After PCR amplification, 1.5% agarose gel electrophoresis was performed, and then the amplified product was recovered by cutting the gel under ultraviolet light, followed by TA cloning and sequencing, and then the amplified products of primers B1, B2 and B3 were spliced to finally obtain wheat BAS1 The cDNA sequence (Seq ID No.2) of the gene is 846bp in size, and the products amplified by primers G1 and G2 are spliced to finally obtain the complete sequence of the wheat BAS1 gene (Seq ID No.1) in size of 2856bp.

Among them, the amplification system used in the PCR reaction is calculated as 10 μl: template DNA 100ng, 10μM primers F and R 0.2μl, 2.5mM dNTP 0.8μl, 5U/μl Taq DNA polymerase ^0.12μl , 10 ×PCR reaction buffer 1.0 μl, make up to 10 μl with ddH ₂ O.

The amplification program of the PCR reaction was: 94°C for 5 minutes; 94°C for 50 seconds, 58°C for 50 seconds, 72°C for 3 minutes, 36 cycles; 72°C for 10 minutes. Store at 4°C.

In the present embodiment, the SDS-Tris saturated phenol method is used to extract the genome DNA of wheat leaves, and the specific method is as follows:

(1) Take 2g of fresh young leaves, grind them into fine powder with liquid nitrogen, and place them in a 2ml sterilized centrifuge tube.

(2) Add 1.2ml DNA extraction buffer (200mM Tris-Cl, pH8.0, 250mM NaCl, 25mM EDTA, pH8.0, 0.5% SDS, 2% β-ME, mix well, add β-ME freshly before use) .

(3) 55°C water bath for 45 minutes, with intermittent shaking during the period to fully extract.

(4) Centrifuge at 12000 rpm for 10 min at room temperature.

(5) Transfer the supernatant to a new 2ml sterilized centrifuge tube, add an equal volume of pre-cooled Tris saturated phenol/chloroform iso/amyl alcohol (volume ratio 25:24:1), mix by inversion on ice for 15min , intermittently oscillating.

(6) Centrifuge at 12000 rpm for 10 min at room temperature.

(7) Transfer the supernatant to a new 2ml sterilized centrifuge tube, and repeat steps (5) and (6) to fully remove the protein.

(8) Transfer the supernatant to a new 1.5ml sterilized centrifuge tube, add 0.6 times the volume of isopropanol (300 μl) and 0.1 times the volume of 50 μl of 5M NaAC solution with a pH of 5.2, mix well and place on ice Let stand for 17min.

(9) White flocculent DNA precipitate appears, centrifuge at 10000rpm for 10min at 4°C.

(10) Discard the supernatant, add pre-cooled 70% ethanol to rinse twice, then rinse once with absolute ethanol, dry at room temperature, add 100 μl 1×TE buffer containing 2 μl 10mg/ml RNase enzyme (or double distilled water) dissolved overnight and diluted 10 times for later use.

(11) Detect the quality and concentration of the extracted wheat genomic DNA with 1% agarose.

The preparation method of wheat cDNA is as follows:

1. Use the TaKaRa kit (Code NO.9769) to extract total RNA from wheat leaves, and follow the instructions of the kit.

2. Using the reverse transcription reaction kit (Code NO.6210A) of TaKaRa Company to synthesize the first strand of cDNA. Methods as below:

(1) Prepare the reaction mixture in the Microtube according to Table 2.

Table 2 reaction mixture

(2) After keeping warm at 65°C for 5 minutes, cool rapidly on ice.

(3) Prepare the reverse transcription reaction solution according to Table 3 in the above-mentioned Microtube tube.

Table 3 reverse transcription reaction solution

(4) Mix slowly.

(5) Reverse transcription reaction was carried out according to the following conditions: 30°C for 10 minutes, 42°C to 50°C for 30 to 60 minutes, and 95°C for 5 minutes, and then cooled on ice to obtain wheat cDNA.

*Example 2 The development of wheat TaBAS1-2B gene functional marker

According to the gene TaBAS1-2B sequence obtained by cloning in Example 1, a polymorphic Indel marker was developed between the two varieties of 'Jing 411' and 'Hongmangchun 21', and a pair of Indel markers were designed using Primer Premier 5.0 software. After amplifying the primers of the Indel marker and analyzing its effect on the phenotype in 194 wheat natural varieties, a gene TaBAS1-2B co-segregated and closely related to wheat leaf chlorophyll content and grain weight was finally developed. Related feature flags.

The pair of primers used to amplify the Indel marker is:

Upstream primer F: 5'-CGCAGTGCCTGTCGTTTC-3'(Seq ID No.5)

Downstream primer R: 5'-TCACATACTTCTTTCCCAAT-3'(Seq ID No.6)

The Indel marker characteristic band associated with the higher chlorophyll content and higher grain weight of wheat leaves amplified by the primer pair is 494bp, and its nucleotide sequence is as shown in Seq ID No.3; The characteristic band of Indel marker associated with lower chlorophyll content and lower grain weight in wheat leaves is 493bp, and its nucleotide sequence is shown in Seq ID No.4.

Example 3 Correlation between different genotypes of wheat TaBAS1-2B gene, chlorophyll content and grain weight in flag leaves after anthesis

1. Determination of chlorophyll content and thousand-grain weight of wheat at different stages after flowering

Chlorophyll determination: 194 natural varieties of wheat were marked for flowering, and the chlorophyll content of flag leaves was measured at 10, 15, 25, 28 and 31 days after flowering. For each variety, three main stem flag leaves with relatively consistent flowering periods are selected, and the upper, middle, and lower spots of each flag leaf are evenly selected, and the chlorophyll content is measured with a SPAD-502 chlorophyll meter, and finally the average value is taken as a measure Chlorophyll level of flag leaves.

Thousand-grain weight measurement: 1000 whole wheat kernels were randomly selected, repeated twice, weighed with a thousandth-grain balance, and the average value of the two times was recorded as the final thousand-grain weight value.

2. The extraction of genomic DNA from wheat leaves is the same as in Example 1.

3. Amplification of the target product. The system and procedure of the PCR amplification reaction are the same as in Example 1.

After PCR amplification, add denatured double indicator (DNA loading buffer) and denature at 95°C for 10 min on a PCR instrument, then place it on ice immediately, and run electrophoresis on 8% denatured polyacrylamide gel after cooling down to room temperature. Silver staining was then developed. Wheat varieties with lower 1000-kernel weight amplified a target band with a size of 493 bp, while varieties with a higher 1000-kernel weight amplified a target band with a size of 494 bp (Fig. 2).

4. Location of TaBAS1-2B gene and its Indel marker on chromosome

The gene TaBAS1-2B was located using the missing tetrasomy material of wheat 'China Spring', and the result was that the gene and its Indel marker were located on the wheat chromosome 2B (Fig. 3).

5. Genotype analysis of different 1000-grain weight wheat varieties and its correlation with chlorophyll content of post-anthesis flag leaf and 1000-grain weight

In 194 wheat natural varieties, the variance analysis was carried out on the chlorophyll content of the flag leaf after anthesis and the thousand-grain weight of the two genotypes, and the correlation analysis was carried out between the allelic variation of the TaBAS1-2B gene and the chlorophyll content of the flag leaf after anthesis and the thousand-grain weight , the results are shown in Table 4.

Table 4 Significant difference analysis of flag leaf chlorophyll content after anthesis and thousand-grain weight between two genotypes in different wheat varieties

Note: The P values of the difference significance levels are 0.05, 0.01 and 0.001, marked by *, ** and *** respectively; the A genotype is the 494bp band pattern, and the B genotype is the 493bp band pattern.

The results in Table 4 showed that the thousand-grain weight of genotype A (494bp) was significantly higher than that of genotype B (493bp). The allele type of the gene reached a very significant level in the 1000-grain weight of 194 materials.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (5)

1. the Indel for spending rear Flag Leaf Blade chlorophyll content and mass of 1000 kernel related gene TaBAS1-2B to isolate with wheat is marked, It is characterized in that, the primer pair for expanding the Indel labels is：

Sense primer F：5’-CGCAGTGCCTGTCGTTTC-3’

Downstream primer R：5’-TCACATACTTCTTCCCAAT-3’

The primer pair amplifies spend rear Flag Leaf Blade to be marked compared with high chlorophyll content and the relevant Indel of higher mass of 1000 kernel with wheat Note characteristic bands are 494bp, and nucleotide sequence is as shown in Seq ID No.3；The primer pair amplifies spend rear flag with wheat The relatively low chlorophyll content of leaf blade and the relevant Indel marker characteristics band of relatively low mass of 1000 kernel are 493bp, and nucleotide sequence is such as Shown in Seq ID No.4.

2. the boot leaf after screening is spent of Indel labels described in claim 1 has small compared with high chlorophyll content and higher mass of 1000 kernel Application in wheat germ plasm resource, which is characterized in that include the following steps：

1）Extract the genomic DNA of plant to be measured；

2）Using the genomic DNA of plant to be measured as template, using primers F described in claim 1 and R, pcr amplification reaction is carried out；

3）Detect pcr amplification product, if it is possible to amplify the characteristic bands of the nucleotide sequence as shown in Seq ID No.3, then Plant to be measured is the wheat resource for spending rear Flag Leaf Blade to have compared with high chlorophyll content and higher mass of 1000 kernel.

3. application according to claim 2, which is characterized in that step 2）The amplification system that middle PCR reactions use is with 10 μ l It is calculated as：Template DNA 100ng, 10 μM of primers Fs and each 0.8 μ l, 5U/ μ l Taq archaeal dna polymerases of 0.2 μ l, 2.5mM dNTP of R 0.12 μ l, Mg containing 25mM²⁺10 × PCR reaction buffers 1.0 μ l, ddH₂O complements to 10 μ l.

4. application according to claim 2, which is characterized in that step 2）Middle PCR reacts the amplification program used：94℃ 5 minutes；94 DEG C 50 seconds, 58 DEG C 50 seconds, 72 DEG C 3 minutes, 36 cycle；72 DEG C 10 minutes.

5. application according to claim 2, which is characterized in that step 3）It is middle to use 6% denaturing polyacrylamide gel electrophoresis Pcr amplification product is detected, then silver staining develops the color.