Promoter mutant that regulates photosynthetic efficiency of maize leaves, molecular marker, and use of promoter mutant and molecular marker

Abstract

Provided are a promoter mutant that regulates the photosynthetic efficiency of maize leaves, a molecular marker, and the use of the promoter mutant and the molecular marker. The mutant has a nucleotide sequence as shown in SEQ ID NO: 1, and the mutant is obtained by means of inserting an 11.2 kb large fragment into a BSD gene promoter. Further provided is a molecular marker closely linked to the insertion of the 11.2 kb large fragment into the BSD gene promoter. The molecular marker is located at 158317719 bp of the BSD gene promoter, with an allelic variant base of A / G, wherein a maize cross-bred line with the large-fragment insertion has base A at said locus, while a maize cross-bred line without the large-fragment insertion has base G at said locus. Further provided are PCR primers and PCR detection kit for detecting the molecular marker, and the use thereof in terms of improving photosynthetic efficiency traits in plants, breeding new plant varieties, etc.

Description

Promoter mutant for regulating photosynthetic efficiency of corn leaf, molecular marker and application thereof TECHNICAL FIELD

[0001] The present application relates to a promoter mutant and a molecular marker associated with the mutation thereof, in particular to a promoter mutant for regulating photosynthetic efficiency of corn leaf and a molecular marker associated with the mutation and application thereof, and belongs to the field of corn promoter mutant and application thereof. BACKGROUND

[0002] Corn has become the largest area of planting and the highest yield of grain in the world, and it is of great significance to improve its photosynthetic efficiency and thus the yield, so its growth and development rule has always been the focus and hotspot of scientific research.

[0003] Plants produce carbohydrates necessary for growth and development through photosynthesis. Plants can be divided into C3 plants, C4 plants and CAM plants according to their photosynthetic types, and C4 plants (such as corn) have higher photosynthetic efficiency than C3 plants. Corn, as a C4 plant, has evolved from C3 plants for a long time, and has also evolved a unique C4 photosynthetic pathway. The CO2 concentration mechanism at the Rubisco reaction site can offset most of the photorespiration. This CO2 concentration mechanism, together with the change of leaf tissue structure, enables C4 plants to have high photosynthetic rate. Corn leaves have two types of morphologically and functionally different photosynthetic cells, i.e. bundle sheath cells and mesophyll cells, which form a unique wreath structure of corn type C4 plants. The photosynthetic enzymes existing in these two types of cells also have great differences: phosphoenolpyruvate carboxylase (PEPC) is specifically located in mesophyll cells, while ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco enzyme) necessary for the Calvin cycle is specifically located in bundle sheath cells. In C4 plants, CO2 is first fixed by PEPC in mesophyll cells to form a four-carbon sugar oxaloacetic acid, which is then transported to adjacent bundle sheath cells in the form of malate and decarboxylated to generate CO2, which is then re-fixed by Rubisco into the Calvin cycle to form carbohydrates to meet the needs of plant growth and development. It is this unique CO2 concentration mechanism that enables C4 plants such as corn to maintain high photosynthetic efficiency regardless of the concentration of O2 in the atmosphere. The abundant photosynthetic enzyme Rubisco in plants is a key enzyme for fixing CO2 in the process of photosynthesis, and has the functions of catalyzing carboxylation and oxidation. Catalyzing photosynthesis or photorespiration depends on the ratio of CO2 / O2 concentration. In corn, Rubisco is mainly located in the intercellular space of bundle sheath cells, so the concentration of CO2 in bundle sheath cells affects the function of Rubisco.

[0004] Maize is a typical C4 plant, which has a unique ring structure and carbon dioxide concentration mechanism, and thus has a high photosynthetic efficiency. The BSD2 (bundle sheath defective 2) gene is mainly expressed in the vascular sheath cells of the ring structure, and the deletion of the BSD2 gene will cause the chloroplasts in the vascular sheath cells at the third leaf base and the leaf tip to be abnormal, with obvious swelling signs, and even the chloroplasts at the leaf tip are ruptured, and the degree of chloroplast swelling and rupture is increased with time. The soluble protein and chlorophyll of the mutant at the seedling stage are reduced, the expression amount of the large subunit and the small subunit of the key enzyme Rubisco enzyme of photosynthesis in the mutant is very low, and even the presence of the Rubisco protein cannot be detected, which causes the seedlings of the homozygous mutant to be yellow, the net photosynthetic rate to be significantly reduced, and the intercellular CO2 concentration to be significantly increased, and finally the seedlings are dead, which indicates that the mutation of the BSD2 gene significantly affects the photosynthesis pathway of maize. SUMMARY

[0005] One of the purposes of the present application is to provide a mutant of the BSD gene promoter for regulating the photosynthetic efficiency of maize leaves.

[0006] The second purpose of the present application is to provide a molecular marker site associated with the mutant of the BSD gene promoter for regulating the photosynthetic efficiency of maize leaves.

[0007] The third purpose of the present application is to apply the mutant of the BSD gene promoter for regulating the photosynthetic efficiency of maize leaves or to apply the molecular marker associated with the mutant to detect whether the photosynthetic efficiency of maize leaves is high or not.

[0008] The above purposes of the present application are achieved by the following technical solutions.

[0009] In one aspect of the present application, a mutant of the BSD gene promoter for regulating the photosynthetic efficiency of maize leaves is provided, wherein an 11.2kb large fragment is inserted into the BSD gene promoter, and the nucleotide sequence of the mutant is shown in SEQ ID No. 1, wherein the insertion sequence of the 11.2kb large fragment is the nucleotide sequence between positions 671-11901 of the nucleotide sequence shown in SEQ ID No. 1.

[0010] The present application further provides a recombinant plant expression vector containing the mutant of the promoter and a host cell containing the recombinant plant expression vector.

[0011] The mutant of the promoter of the present application is operably linked to the heterologous DNA sequence to be transcribed, so as to obtain a recombinant plant expression vector for expressing the heterologous DNA sequence in plants.

[0012] The recombinant plant expression vector can further comprise a selection marker gene.

[0013] The recombinant plant expression vector constructed in the application can be introduced into cells, tissues or organs of target plants by any plant transformation method to obtain transformants; and the transformants are regenerated into complete plants, clones or offspring thereof by plant tissue culture method; the transformation method includes Agrobacterium-mediated transformation, protoplast transformation, Ti plasmid, Ri plasmid, plant virus vector, microinjection, electroporation, microprojectile bombardment and the like; the target plants include monocotyledonous plants and dicotyledonous plants; preferably, the target plants are Gramineae plants, for example, can be corn, rice, barley, wheat, sorghum and the like, and preferably corn.

[0014] Another aspect of the application is to provide a molecular marker closely associated with the insertion of the 11.2 kb large fragment into the BSD gene promoter, which is located at 158317719 bp of the BSD gene promoter, and the allelic variation base is A / G; the base at the site of the corn inbred line with the insertion of the 11.2 kb large fragment is A, and the base at the site of the corn inbred line without the insertion of the 11.2 kb large fragment is G; wherein the BSD gene is optionally any one of the following versions of BSD genes: V3 version number GRMZM2G062788; V4 version number Zm00001d030786; V5 version number Zm00001eb029690.

[0015] Another aspect of the application is to provide a PCR primer for amplifying the above-mentioned SNP molecular marker, which is selected from any one of the PCR primers described in (1)-(2) below:

[0016] Primer group (1) is composed of the upstream primer shown in SEQ ID No. 2 and the downstream primer shown in SEQ ID No. 3;

[0017] Primer group (2) is composed of the upstream primer shown in SEQ ID No. 4 and the downstream primer shown in SEQ ID No. 5.

[0018] Still another aspect of the application is to provide a method for detecting whether the 11.2 kb large fragment is inserted into the BSD gene promoter of corn by using the PCR primer, which comprises the following steps:

[0019] (1) extracting the DNA of the corn genome to be detected;

[0020] (2) establishing a PCR amplification system by using the extracted DNA of the corn genome as a template and using primer group (1) or primer group (2) as PCR primers to perform PCR amplification;

[0021] (3) wherein, if PCR amplification is carried out with the primer set (1) as PCR primers, a product with a length of 2059 bp is amplified, indicating that the 11.2 kb large fragment insertion exists in the BSD gene promoter of the sample to be detected; if PCR amplification is carried out with the primer set (2) as PCR primers, if a product with a length of 2037 bp is amplified, indicating that the 11.2 kb large fragment insertion exists in the BSD gene promoter of the sample to be detected.

[0022] Still another aspect of the present application provides a method for detecting the photosynthetic efficiency of corn leaves by using the PCR primers, comprising:

[0023] (1) extracting the DNA of the corn genome to be detected;

[0024] (2) using the extracted DNA of the corn genome as a template, and using the primer set (1) or the primer set (2) as PCR primers to establish a PCR amplification system for PCR amplification;

[0025] (3) wherein, if PCR amplification is carried out with the primer set (1) as PCR primers, a product with a length of 2059 bp is amplified, indicating that the photosynthetic efficiency of the corn leaves to be detected is low; if PCR amplification is carried out with the primer set (2) as PCR primers, if a product with a length of 2037 bp is amplified, indicating that the photosynthetic efficiency of the corn leaves to be detected is low.

[0026] In a preferred embodiment of the present application, the PCR amplification system in step (2) is: 1.5 μL of PCR upstream primer, 1.5 μL of PCR downstream primer, 5 μL of template DNA, 1 μL of KOD FX, 10 μL of dNTPs, 25 μL of PCR buffer, and 6 μL of ddH2O; and the PCR amplification program is: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 10 s, 60℃ annealing for 30 s, and 68℃ extension for 2 min, 30-35 cycles; 68℃ for 10 min, and 25℃ for min.

[0027] Still another aspect of the present application provides a PCR detection kit for detecting whether the 11.2 kb large fragment insertion exists in the BSD gene promoter of corn or the photosynthetic efficiency of leaves is high or low, comprising: 1.5 μL of PCR upstream primer, 1.5 μL of PCR downstream primer, KOD FX, dNTPs, PCR buffer, and ddH2O; wherein the PCR upstream primer and the PCR downstream primer consist of the upstream primer shown in SEQ ID No. 2 and the downstream primer shown in SEQ ID No. 3; or consist of the upstream primer shown in SEQ ID No. 4 and the downstream primer shown in SEQ ID No. 5.

[0028] The BSD gene promoter mutant for regulating photosynthetic efficiency of corn leaves provided by the application and the SNP molecular marker associated with the insert fragment of the mutant have application prospects in improving plant photosynthetic efficiency traits, cultivating new plant varieties and the like.

[0029] Definitions of terms involved in the application

[0030] The term "host cell" or "recombinant host cell" means a cell which contains a polynucleotide of the application, regardless of the method by which it was inserted into the recombinant host cell, e.g., direct uptake, transduction, f-mating, or other means known in the art. The exogenous polynucleotide can remain, for example, in a non-integrated vector, or can be integrated into the host genome.

[0031] The term "promoter" refers to a recognition site for RNA polymerase and other factors necessary for proper transcription initiation, present upstream from the coding sequence of a gene of interest, which initiates or directs transcription of a gene of interest into mRNA.

[0032] The term "selectable marker gene": expression of this gene in plant cells confers a selection advantage to the cell. The selection advantage possessed by cells transformed with these selectable marker genes can be due to their ability to grow in the presence of a negative selection agent (e.g., an antibiotic or herbicide) as compared to non-transformed cells. Selectable marker genes also refer to combinations of genes whose expression in plant cells confers both negative and positive selection advantages to the cell.

[0033] The term "operably linked" refers to functional linkage between two or more elements, e.g., elements are in a functional relationship to one another. An operable linkage of elements means that the elements are linked in such a way as to permit them to function together as a unit.

[0034] The term "transformation": a method of introducing a heterologous DNA sequence into a host cell or organism.

[0035] The term "expression": transcription and / or translation of an endogenous gene or transgene in a plant cell.

[0036] The term "plant expression vector": one or more DNA vectors used to effect transformation of a plant; these vectors are often referred to in the art as binary vectors. Binary vectors, along with vectors having helper plasmids, are most commonly used for Agrobacterium-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, a selectable marker engineered to be capable of expression in plant cells, a heterologous DNA sequence to be transcribed, etc. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a schematic diagram of a structural variation of 11.2 kb in length of the BSD2 promoter region of corn; the insertion of the 11.2 kb large fragment is closely associated with a SNP site (A / G).

[0038] Figure 2 is a result of a large fragment structural variation in the BSD2 promoter region directly affecting the transcript level of the BSD2 gene.

[0039] Figure 3 is a result of comparative analysis between the large fragment insertion variation existing in the BSD2 gene promoter region and the leaf photosynthetic efficiency without the large fragment insertion. DETAILED DESCRIPTION

[0040] The advantages and features of the present application will become more apparent with the following detailed description. However, the following examples are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the present application without departing from the spirit and scope of the present application, and such modifications and substitutions fall within the scope of the present application.

[0041] Example 1 Identification of large fragment structural variation in the BSD2 gene promoter region and SNP molecular marker site closely associated with the large fragment insertion

[0042] The BSD2 gene (Gene ID: V3 version number GRMZM2G062788; V4 version number Zm00001d030786; V5 version number Zm00001eb029690).

[0043] Through genome sequencing of 350 representative inbred lines of different breeding years of corn in China and the United States, it was found that a structural variation with a length of 11.2 kb was found in the BSD2 promoter region, and the 350 representative inbred lines could be divided into large fragment insertion (YES) or no (NO), and there was a significant cis-eQTL, and there was a SNP site (A / G) at the position 158317719 bp in the promoter region, which was closely associated with the large fragment, and the corn inbred lines with large fragment insertion (YES) were A at the site, and the corn inbred lines with large fragment deletion were G at the site (Figure 1).

[0044] Through transcriptome data analysis of 137 corn inbred lines in China, it was found that the average expression level of BSD2 in corn inbred lines with large fragment insertion (YES, A type) was significantly lower than that in corn inbred lines with large fragment deletion (NO, G type), indicating that the structural variation regulates the expression level of BSD2 (Figure 2).

[0045] Test Example 1 Correlation analysis between large fragment structural variation in the BSD2 gene promoter region and leaf photosynthetic efficiency

[0046] In order to verify the correlation between the genotype and phenotype of the large fragment structural variation (insertion or deletion) in the promoter region of the BSD2 gene, 83 corn inbred lines with 11.2 kb large fragment insertion (YES) in the promoter region of the BSD2 gene and 165 corn inbred lines without 11.2 kb large fragment insertion (NO) in the promoter region of the BSD2 gene were used to investigate photosynthetic parameters. The results showed that the transpiration rate (E), net photosynthetic rate (A), CO2 stomatal conductance (gsw), and photochemical efficiency (PSII) of the corn inbred lines with 11.2 kb large fragment insertion (YES) in the promoter region of the BSD2 gene were significantly lower than those of the corn inbred lines without 11.2 kb large fragment insertion (NO) in the promoter region of the BSD2 gene, indicating that the 11.2 kb large fragment insertion in the promoter region of the BSD2 gene led to a significant decrease in the photosynthetic efficiency of the plant leaves (Figure 3).

[0047] Test Example 2: Test for detecting whether a large fragment is inserted in the promoter region of the BSD2 gene of corn according to the design of PCR detection primers

[0048] The primer pair SV-F1: 5'-GCTTTACAAAACACTTAGAGAAGT-3' (SEQ ID No. 2) and SV-R1: 5'-ATGCGTCCTTAACACAAGGCGCTG-3' (SEQ ID No. 3) can be used for amplification, with a length of 2059 bp (SEQ ID No. 6), and identification by agarose electrophoresis and DNA sequencing. If a product with a length of 2059 bp is amplified, it indicates that there is an 11.2 kb large fragment insertion in the promoter of the BSD gene of the sample to be detected.

[0049] The primer pair SV-F2: 5'-GTCTCGTGCGTCGAAGGTTAGCG-3' (SEQ ID No. 4) and SV-R2: 5'-CCGGTTATGTTTGCAAATAGGCAC-3' (SEQ ID No. 5) can also be used for amplification, with a length of 2037 bp (SEQ ID No. 7), and identification by agarose electrophoresis and DNA sequencing. If a product with a length of 2037 bp is amplified, it indicates that there is an 11.2 kb large fragment insertion in the promoter of the BSD gene of the sample to be detected.

[0050] In the amplification, the PCR reaction components are shown in Table 1:

[0051] Table 1: PCR reaction components

[0052] The PCR program is as follows:

Claims

1. A mutant of the BSD gene promoter that regulates the photosynthetic efficiency of corn leaves, characterized in that: The nucleotide sequence of the mutant is shown in SEQ ID No.

1. The mutant has an 11.2 kb large fragment inserted into the BSD gene promoter. The insertion sequence of the 11.2 kb large fragment is the nucleotide sequence between positions 671 and 11901 of nucleotides shown in SEQ ID No.

1.

2. A recombinant plant expression vector comprising the mutant of the BSD gene promoter according to claim 1.

3. A recombinant host cell containing the recombinant plant expression vector according to claim 2.

4. A molecular marker closely associated with the insertion of an 11.2 kb large fragment in the mutant of the BSD gene promoter according to claim 1, characterized in that: The molecular marker is located at 158317719 bp of the BSD gene promoter, the allelic variation base is A / G, the base at this site of the corn inbred line with the 11.2 kb large fragment inserted is A, and the base at this site of the corn inbred line without the 11.2 kb large fragment inserted is G; wherein, the BSD gene can be any one of the following version numbers of the BSD gene: V3 version number GRMZM2G062788; V4 version number Zm00001d030786; V5 version number Zm00001eb029690.

5. PCR primers for detecting the molecular marker according to claim 4.

6. The PCR primer according to claim 5, characterized in that The PCR primer is selected from any one of the following PCR primers (1)-(2): Primer set (1): consisting of the upstream and downstream primers shown in SEQ ID No. 2 and SEQ ID No. 3; Primer set (2): consists of the upstream and downstream primers shown in SEQ ID No. 4 and SEQ ID No.

5.

7. A method for detecting whether there is an 11.2 kb large fragment inserted in the maize BSD gene promoter using the PCR primers according to claim 6, characterized in that: include: (1) extracting DNA from the corn genome to be tested; (2) using the extracted corn genomic DNA as a template and primer set (1) or primer set (2) as PCR primers to establish a PCR amplification system for PCR amplification; (3) Among them, if primer set (1) is used as a PCR primer for PCR amplification, a product with a length of 2059 bp is obtained, indicating that a large fragment of 11.2 kb is inserted in the BSD gene promoter of the sample to be tested; if primer set (2) is used as a PCR primer for PCR amplification, if a product with a length of 2037 bp is obtained, indicating that a large fragment of 11.2 kb is inserted in the BSD gene promoter of the sample to be tested.

8. A method for detecting the photosynthetic efficiency of corn leaves using the PCR primers according to claim 6, characterized in that: include: (1) extracting DNA from the corn genome to be tested; (2) using the extracted corn genomic DNA as a template and primer set (1) or primer set (2) as PCR primers to establish a PCR amplification system for PCR amplification; (3) If primer set (1) is used as a PCR primer for PCR amplification, a product with a length of 2059 bp is obtained, indicating that the photosynthetic efficiency of the corn leaf to be tested is low; if primer set (2) is used as a PCR primer for PCR amplification, a product with a length of 2037 bp is obtained, indicating that the photosynthetic efficiency of the corn leaf to be tested is low.

9. A PCR detection kit for detecting the photosynthetic efficiency of corn, comprising: 1.5 μL of PCR upstream primer, 1.5 μL of PCR downstream primer, KOD FX, dNTPs, PCR buffer, ddH2O; characterized in that the PCR upstream primer and PCR downstream primer are the PCR primers according to claim 5 or 6.

10. Use of the mutant of the BSD gene promoter according to claim 1, the molecular marker according to claim 4, and the PCR primer according to claim 5 or 6 in improving plant photosynthetic efficiency traits or breeding new plant varieties.

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