Protein PCE1 regulates plant height and grain weight in sorghum

Abstract

The present invention discloses the application of protein PCE1 in regulating plant height and grain weight of sorghum. The protein PCE1 has an amino acid sequence as shown in SEQ ID NO.1. Compared with WT, the plant height and grain weight of the obtained PCE1 protein single base deletion mutant material pce1 are significantly reduced. The specific fragment of the gene encoding the PCE1 protein is subjected to transgenic silencing to obtain T2 generation PCE1 gene silenced sorghum. Compared with the transgenic receptor material P898012, the plant height and grain weight of the T2 generation PCE1 gene silenced sorghum are significantly reduced. Therefore, reducing the expression amount of protein PCE1 in sorghum can regulate the plant height and grain weight of sorghum. The present invention has important application value in regulating the plant height and grain weight of sorghum.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of protein PCE1 in regulating plant height and grain weight of sorghum. Background Art

[0002] Sorghum (Sorghum bicolor L.Moench) is an annual C4 herbaceous plant belonging to the genus Sorghum of the Poaceae family. It originated in Africa and is one of the earliest important cereal crops cultivated by humans. It is also the fifth largest food crop after rice, corn, wheat and barley. Sorghum has high light energy utilization rate, strong adaptability, and multiple resistances such as drought resistance, waterlogging resistance, cold resistance, and salt-alkali resistance. It is widely distributed in arid, semi-arid, low-lying, waterlogged and saline-alkali areas. At the same time, sorghum has a wide range of uses, including food, feed, brewing, bioenergy and chemical materials. It is a feed crop and green energy crop with development potential.

[0003] With the increasing demand for freshwater resources, the reduction of cultivated land and the severe environmental conditions of global warming, drought-resistant and stress-resistant crop sorghum is becoming increasingly important in supplying the world's growing food demand. The cultivation and planting of high-yield sorghum varieties will have a profound impact on the world's food security. Plant height and grain yield are important agronomic traits that affect sorghum biomass and yield. Therefore, cloning related genes in sorghum and applying them to sorghum breeding and production has important theoretical significance and practical value. It is also an effective way to regulate sorghum plant type and grain weight. In recent years, the cloning of sorghum grain weight genes has progressed slowly, and there is an urgent need to provide molecules that affect sorghum grain weight. Summary of the invention

[0004] The technical problem to be solved by the invention is how to regulate the plant height and grain weight of sorghum.

[0005] In order to solve the above technical problems, the present invention first provides the application of protein PCE1 in regulating sorghum plant height and grain weight;

[0006] The protein PCE1 may be a1) or a2) or a3):

[0007] a1) the amino acid sequence is the protein shown in SEQ ID NO.1;

[0008] a2) a fusion protein obtained by connecting a tag to the N-terminus and / or the C-terminus of the protein shown in SEQ ID NO.1;

[0009] a3) A protein related to sorghum plant height and / or grain weight obtained by replacing and / or deleting and / or adding one or more amino acids of the amino acids shown in SEQ ID NO.1.

[0010] Among them, SEQ ID NO.1 consists of 139 amino acids.

[0011] Preferably, the tag described in a2) is as shown in Table 1, and the tag is connected to facilitate the purification of the protein in a1).

[0012] Table 1 Tag sequences

[0013] Label Residue sequence Poly-Arg 5-6 (usually 5) RRRRR FLAG 8 DYKDDDDK Strep-tag II 8 WSHQ c-myc 10 EQKLISEEDL

[0014] Preferably, in the protein described in a3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0015] Preferably, the protein in a3) above is artificially synthesized.

[0016] Preferably, the protein in a3) above is first synthesized by its coding gene and then obtained by biological expression.

[0017] The coding gene of the protein in a3) above can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID NO.2, and / or performing missense mutation of one or several base pairs, and / or connecting the coding sequence of the tag shown in Table 1 to its 5' end and / or 3' end.

[0018] The use of the nucleic acid molecule encoding the protein PCE1 in regulating sorghum plant height and grain weight also falls within the protection scope of the present invention.

[0019] The nucleic acid molecule encoding the protein PCE1 may be a DNA molecule as shown in b1) or b2) or b3) or b4) below:

[0020] b1) a DNA molecule whose coding region is shown in SEQ ID NO.1;

[0021] b2) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.2;

[0022] b3) a DNA molecule that has 75% or more identity with the nucleotide sequence defined in b1) or b2) and encodes the protein PCE1;

[0023] b4) A DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in b1) or b2) and encodes the protein PCE1.

[0024] Wherein, the nucleic acid molecule is DNA, such as cDNA, genomic DNA or recombinant DNA;

[0025] Preferably, the nucleic acid molecule is RNA, such as mRNA or hnRNA.

[0026] Among them, SEQ ID NO.2 in the sequence listing consists of 832 nucleotides, and the nucleotides of SEQ ID NO.2 in the sequence listing encode the amino acid sequence shown in SEQ ID NO.1 in the sequence listing.

[0027] A person skilled in the art can easily mutate the nucleotide sequence encoding the protein PCE1 of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of the protein PCE1 isolated by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein PCE1.

[0028] As used herein, "identity" refers to sequence similarity with a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, or 80% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein PCE1 composed of the amino acid sequence shown in SEQ ID NO.1 of the coding sequence table of the present invention. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0029] The present invention also protects a method for cultivating transgenic sorghum, comprising the following steps: inhibiting the expression level and / or activity of any of the above-mentioned proteins PCE1 in the starting sorghum to obtain transgenic sorghum; compared with the starting sorghum, the plant height and grain weight of the transgenic sorghum are reduced.

[0030] In the above method, suppressing the expression level and / or activity of any of the above-mentioned proteins PCE1 in the starting sorghum is achieved by introducing a substance that suppresses the expression of a nucleic acid molecule encoding the protein PCE1 into the starting sorghum.

[0031] The substance that inhibits the expression of the nucleic acid molecule encoding protein PCE1 is a specific DNA molecule, an expression cassette containing the specific DNA molecule, or a recombinant plasmid containing the specific DNA molecule;

[0032] The specific DNA molecule includes a sense segment, an antisense segment and a spacer segment between them;

[0033] The positive fragment is the DNA molecule shown in SEQ ID NO.2 from 335 to 518;

[0034] The antisense fragment is the reverse complementary sequence of the DNA molecule shown in SEQ ID NO.2 from 385 to 552.

[0035] In the above method, suppressing the expression level and / or activity of any of the above-mentioned proteins PCE1 in the starting sorghum is achieved by introducing the interference vector RNAi-PCE1 into the starting sorghum.

[0036] The interference vector RNAi-PCE1 is a recombinant plasmid obtained by replacing the small DNA fragment between the restriction endonucleases BamHI and XbaI of the interference vector pFGC5941 with the reverse complementary sequence of the DNA molecule shown in SEQ ID NO.2 from 385 to 552, and replacing the small DNA fragment between the restriction endonucleases AscI and SmiI with the DNA molecule shown in SEQ ID NO.2 from 335 to 518.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] Experiments have shown that the interference vector RNAi-PCE1 was introduced into the starting sorghum P898012 to obtain T2 generation PCE1 gene silenced sorghum lines RNAi-1 and RNAi-2. Compared with the starting sorghum P898012, the plant height of RNAi-1 and RNAi-2 sorghums was reduced, the ears were shortened, the grains were shortened and narrowed, and the grain weight was reduced. It can be seen that reducing the expression of protein PCE1 in P898012 can regulate the plant type and grain weight of sorghum. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The figure is a phenotypic comparison between the mutant pce1 and the wild type, where A is the comparison of plant height phenotype, B is the comparison of ear length phenotype, C is the comparison of ear grain phenotype, D is the comparison of 100 grains, E is the comparison of grain length phenotype, F is the comparison of grain width phenotype, G is the comparison of plant height, H is the comparison of ear length, I is the comparison of 100-grain weight, J is the comparison of grain length, and K is the comparison of grain width;

[0040] Figure 2 is the cloning of PCE1 gene, where A is the distribution of Δ(SNP-index) on chromosome, and B is the location of PCE1;

[0041] Figure 3 Real-time fluorescence quantitative PCR was used to detect the relative expression level of PCE1 gene in T2 generation PCE1 gene silenced sorghum lines;

[0042] Figure 4This is the phenotypic identification of sorghum with silenced PCE1 gene, where A is the comparison of plant height phenotype, B is the comparison of ear length phenotype, C is the comparison of grain length phenotype, D is the comparison of grain width phenotype, E is the comparison of plant height, F is the comparison of ear length, G is the comparison of 100-grain weight, H is the comparison of grain length, and I is the comparison of grain width. DETAILED DESCRIPTION

[0043] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0044] Example 1. Creation, screening and phenotypic identification of mutant pce1

[0045] To obtain the sorghum mutant pce1, the sorghum variety Shangzhuang was used as the base material, and EMS with a concentration of 0.25% was used for mutagenesis for 12 hours. The sorghum mutant library was constructed by screening mutants from the M2 generation mutagenesis population. The M3 generation was planted according to the line and the pce1 mutant with stable phenotype and non-segregation was screened. Compared with Shangzhuang, the pce1 mutant had a lower plant height, shorter panicle length, shorter and narrower grains, and lower grain weight. Figure 1 shown.

[0046] Example 2: Cloning of PCE1 gene

[0047] The pce1 mutant material was bagged and selfed for two generations, and then backcrossed with Shangzhuang and selfed, resulting in a genetic segregation population consisting of 271 plants. Twenty typical plants of the Shangzhuang phenotype and the pce1 mutant were selected from the segregation population, and the wild progeny pool (QUN-WT-pool) and mutant progeny pool (QUN-pce1-pool) were constructed with equal amounts of genomic DNA. At the same time, the wild parent pool (WT-pool) and mutant parent pool (pce1-pool) were constructed for Shangzhuang and mutant pce1, respectively. After completing the above steps, high-throughput sequencing technology was used to perform high-throughput resequencing on the four mixed pools, and the SNP-index of the two extreme trait progeny pools was subtracted to obtain the Δ(SNP-index) distribution between the two progeny. Referring to the Mut-map positioning analysis method, a confidence interval with a confidence level of 0.01 was selected. Analysis showed that the mutation site SV1-1 was located in the coding region of the gene Sobic.001G034700, causing an adenine (A) deletion mutation in the third exon of the Sobic.001G034700 gene, resulting in premature termination of protein translation. The first-generation sequencing technology was used to perform PCR amplification and sequencing on the mixed pool single strains to verify the accuracy of the mutation site, such as Figure 2 shown.

[0048] Example 3: Obtaining the full-length sequence of PCE1 gene

[0049] 1. Extraction of Whole-Genome RNA

[0050] The total RNA of the young panicles of sorghum variety Shangzhuang was extracted using RNAprep Pure Plant Total RNA Extraction Kit (TIANGEN), and then reverse transcribed using SuperScriptII reverse transcriptase (Invitrogen, Cat no. 18064-014) to obtain the total cDNA of Shangzhuang.

[0051] 2. RACE experiment of PCE1 gene

[0052] Using Clontech's RACE5' / 3'Kit, which uses The technology can start with polyA+ or total RNA, and the synthesized first-strand cDNA can be directly used for 5' and 3' RACE-PCR reactions without the need for Adaptor connection, and the full-length cDNA sequence can be obtained sensitively, efficiently and specifically. The specific steps are as follows:

[0053] 1. Synthesize cDNA for RACE experiment (5'or3'-RACE-ReadycDNA).

[0054] 2. Prepare 5' and 3' RACE reaction systems according to the kit. The Tm value of GSP primer is >70℃, so the Touchdown program is used for amplification.

[0055] 3. Take 5 μL of the reaction product for agarose gel electrophoresis detection, observe the presence and position of the bands, and confirm the PCR amplification product.

[0056] 4. Purify the PCR products of 5' and 3' RACE respectively, ligate them into pMD18-T vector, and select positive clones for sequencing.

[0057] 3. Full-length sequence amplification of PCE1 gene

[0058] The cDNA obtained in step 2 was used as a template, and primer 1 was used to amplify the full-length sequence of the PCE1 gene. The PCR product was recovered and purified to obtain a DNA fragment of about 832 bp. The sequencing results showed that the nucleotide sequence of the recovered fragment was as shown in SEQ ID NO.2, specifically. The DNA fragment shown in SEQ ID NO.2 from the 135th to 554th position from the 5' end is the PCE1 gene (specifically, the coding region of the PCE1 gene). Among them, primer 1 is:

[0059] Primer F: 5'-ACTCGGCACGCAGGCAGCACAG-3', recorded as SEQ ID NO. 3;

[0060] Primer R: 5'-TCATGAATAGGGAACATATCGA-3', recorded as SEQ ID NO.4.

[0061] The PCE1 gene encodes protein PCE1. The amino acid sequence of protein PCE1 is shown in SEQ ID NO.1, specifically MANARSGVAVNDECMLKFGELQSKRLHRFITYKMDDKFKEIVVDQVGDRATSYEDFTNSLPENDCRYAIYDFDFVTAEDVQKSRIFYILWSPDSAKVKSKMLYASSNQKFKSGLNGIQVELQATDASEISIDQIKDRAR.

[0062] Example 4: Acquisition of sorghum with silenced PCE1 gene and its phenotypic identification

[0063] 1. Construction of interference vector RNAi-PCE1

[0064] The expression vector pFGC5941 was used as an empty vector, and the empty vector was double-digested with restriction endonucleases to obtain a linearized vector; specific primers were designed according to the specific segment of the CDS of the PEC1 gene, and the specific primer sequences were modified and processed. The forward fragment and the reverse complementary fragment were amplified using the cDNA of the young spike of Shanghaizhuang as a template, and the forward fragment was first connected to the vector using the homologous recombination method, and the positive plasmid containing the forward fragment was selected, and the reverse complementary fragment was connected to the restriction sites AscI and SmiI of the positive plasmid. The specific operation steps are as follows:

[0065] 1. Use RNAprep Pure Plant Total RNA Extraction Kit (TIANGEN) and SuperScriptII Reverse Transcriptase (Invitrogen, Cat no. 18064-014) to obtain cDNA of Shangzhuang.

[0066] 2. Using the cDNA obtained in step 1 as a template, use a pair of specific primers to perform PCR amplification of the target fragment to obtain PCR product 1.

[0067] Wherein, the sequence after modification of the specific primer 1 is as follows:

[0068] pRNAi-PCE1-1F: 5'-TGGTCAATTTGCAGGTATTTGGATCC CGCAATCTATGATTTTGACT-3' (underlined is the primer sequence), recorded as SEQ ID NO.5;

[0069] pRNAi-PCE1-1R: 5'-TCCCGGGTCTTAATTAACTCTCTAGAAT TTCACTTGCATCTGTAGC -3' (the underline is the primer sequence), recorded as SEQ ID NO.6.

[0070] 3. Double-digest the PCR product 1 obtained by amplification in step 2 with restriction endonucleases AscI and SmiI, and purify and recover the DNA fragment 1 of about 183 bp.

[0071] 4. Use AscI and SmiI to double-digest the pFGC5941 vector and purify and recover the vector backbone 1.

[0072] 5. Connect the DNA fragment 1 purified and recovered in step 3 and the vector backbone 1 digested and recovered in step 4 to obtain an intermediate vector.

[0073] 6. Using the cDNA obtained in step 1 as a template, perform PCR amplification of the target fragment using two pairs of specific primers to obtain PCR product 2.

[0074] The sequence of the modified specific primer 2 is as follows:

[0075] pRNAi-PCE1-2F: 5'-ACATTTACAATTACCATGGGGCGCGCC AGCGTGCCCGATCCTTGATC -3' (underlined is the primer sequence), recorded as SEQ ID NO.7;

[0076] pRNAi-PCE1-2R: 5'-AACATAAGAAATTCTTACACATTTAAAT GGATATTCTATATCCTATGG -3' (the underline is the primer sequence), recorded as SEQ ID NO.8.

[0077] 7. Double digest the PCR product 2 obtained in step 6 with BamHI and XbaI, and purify and recover the DNA fragment 2 of about 167 bp.

[0078] 8. Use BamHI and XbaI to double-digest the intermediate vector obtained in step 5 to purify and recover the vector backbone 2.

[0079] 9. Connect the DNA fragment 2 purified and recovered in step 7 and the vector backbone 2 recovered by enzyme digestion in step 8 to obtain the silencing / or interference vector RNAi-PCE1.

[0080] The constructed RNAi-PCE1 vector was sequenced and analyzed. The results showed that the small DNA fragment between the restriction endonucleases BamHI and XbaI in the pFGC5941 vector was replaced with the reverse complementary sequence of the DNA molecule shown in SEQ ID NO.2 from 385 to 552, and the small DNA fragment between the restriction endonucleases AscI and SmiI was replaced with the DNA molecule shown in SEQ ID NO.2 from 335 to 518, thereby obtaining a recombinant plasmid.

[0081] 2. Obtaining PCE1-silenced sorghum and its phenotypic identification

[0082] The transgenic plants were transformed into the starting sorghum variety P898012 by Agrobacterium infection. The PCE1 gene expression in the T2 transgenic plants was detected using specific primers, and the phenotypes of the phenotypically positive plants were statistically analyzed ( Figure 3 ; Figure 4 ). The specific steps are as follows:

[0083] 1. Introduce the interference vector RNAi-PCE1 into AGL1 to obtain recombinant Agrobacterium AGL1 / RNAi-PCE1.

[0084] 2. AGL1 / RNAi-PCE1 was transformed into P898012 by Agrobacterium infection to obtain T0 transgenic sorghum plants, and T2 positive plants were obtained by continuous self-pollination. Specific primers were used to detect the positive transgenic seedlings of each generation. The detection method was as follows: genomic DNA from the leaves of the seedlings to be tested was extracted and PCR amplified using specific primers.

[0085] Wherein, the specific primer is:

[0086] T-RNAi-PCE1-1F: 5'-TCCTTAAGTAGCATCACACGTG-3', recorded as SEQ ID NO. 9;

[0087] T-RNAi-PCE1-1R: 5'-ATTTCACTTGCATCTGTAGC-3', recorded as SEQ ID NO. 10;

[0088] T-RNAi-PCE1-2F: 5'-ACAGTAGAAAAGGAAGGTGGC-3', recorded as SEQ ID NO. 11;

[0089] T-RNAi-PCE1-2R: 5′-GGATATTCTATATCCTATGG-3′, recorded as SEQ ID NO.12.

[0090] 3. The relative expression level of PCE1 gene in RNAi-1 and RNAi-2 of T2 positive plants was detected by qRT-PCR. The primers for detecting PCE1 gene expression were:

[0091] PCE1-RT-PCR-1F: CGAGTGCATGCTGAAGTTTG, recorded as SEQ ID NO. 13;

[0092] PCE1-RT-PCR-1R: ATTGCGTATCGGCAGTCATT, recorded as SEQ ID NO.14.

[0093] Some test results are as follows Figure 3 Compared with P898012, the relative expression levels of PCE1 gene in the T2 generation PCE1 gene silenced sorghum were significantly down-regulated, and the expression levels of the two T2 generation silenced lines were significantly down-regulated, which were named RNAi-1 and RNAi-2. Figure 3 shown.

[0094] 4. Conventionally plant P898012, RNAi-1 and RNAi-2 transgenic materials in 3 rows, with 20 plants in each row. When the plants mature, statistical analysis is performed on their plant height, ear length, grain length, grain width and 100-grain weight.

[0095] The statistical results are as follows Figure 4 As shown, compared with P898012, the plant height of T2 generation PCE1 gene silenced sorghum (RNAi-1, RNAi-2) was reduced, the grain length was shortened, the grain width was narrowed, and the 100-grain weight was reduced.

[0096] The above results indicate that reducing the expression level of PCE1 protein in sorghum can regulate the plant height and grain weight of sorghum, and regulating grain weight is manifested by regulating grain length, grain width and 100-grain weight.

[0097] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0098] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0099] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of protein PCE1, characterized in that: For S1) or S2): S1) regulate sorghum plant height and grain weight; S2) Cultivating transgenic sorghum with target plant height and grain weight; Wherein, the regulation in S1) is manifested as reducing the plant height and the grain weight by reducing the expression level of PCE1 protein; The protein PCE1 is a1): a1) The amino acid sequence is the protein shown in SEQ ID NO.

1.

2. Use of a nucleic acid molecule encoding the protein PCE1 according to claim 1, characterized in that: For S1) or S2): S1) Regulate sorghum plant height and grain weight; S2) Cultivating transgenic sorghum with target plant height and grain weight; The regulation in S1) is manifested as reducing the plant height and the grain weight by silencing the PCE1 gene.

3. The use according to claim 2, characterized in that: The nucleic acid molecule is a DNA molecule as shown in b1) or b2) below: b1) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.2 from position 135 to position 554; b2) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.

2.

4. The use according to claim 1 or 2, characterized in that: Reducing the plant height and the grain weight is manifested in reduced sorghum plant height, shorter grain length, narrower grain width, and reduced 100-grain weight.

5. Use of a specific DNA molecule, an expression cassette containing the specific DNA molecule, or a recombinant plasmid containing the specific DNA molecule, characterized in that: For S1) or S2): S1) Regulate sorghum plant height and grain weight; S2) Cultivating transgenic sorghum with target plant height and grain weight; Wherein, the regulation in S1) is manifested as reducing the plant height and the grain weight by silencing the PCE1 gene; The specific DNA molecule includes a sense segment, an antisense segment and a spacer segment between them; The sense fragment is the DNA molecule shown in SEQ ID NO.2 from position 335 to position 518 in claim 3; The antisense fragment is the reverse complementary sequence of the DNA molecule shown in SEQ ID NO.2 from position 385 to position 552 in claim 3.

6. The use according to claim 5, characterized in that: Reducing the plant height and the grain weight is manifested in that the plant height of sorghum is reduced, the grain length is shortened, the grain width is narrowed, and the 100-grain weight is reduced.

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