A fertility gene IpCYTc of Idesia polycarpa and its application

Abstract

The present invention relates to the technical field of genetic engineering, and in particular to a Castanopsis affine fertility gene IpCYTc and an application thereof. The Castanopsis affine fertility gene IpCYTc has a nucleotide sequence as shown in SEQ ID NO.1. The present invention utilizes a rice anther-specific promoter to overexpress IpCYTc and genetically transforms it into rice, and a decrease in rice anther fertility can be detected, thereby providing new materials for the application of rice and Castanopsis affine male sterile lines in hybrid seed production.

Description

Technical Field

[0001] The present invention relates to the technical field of gene engineering, and in particular to a Castanopsis affine fertility gene IpCYTc and an application thereof. Background Art

[0002] Castanopsis chinensis is a woody oil-bearing tree species that is currently being promoted on a large scale in my country. It is a dioecious tree species with typical primitive bisexual flower characteristics in the early stages of flower development. As the flower buds develop, the primordium of one sex aborts at a specific stage, thus forming a unisexual flower, in which remnants of the flower organs of the opposite sex can be observed. However, in actual production, the flower primordium of the opposite sex does not degenerate to form bisexual flowers, resulting in nutrient dispersion and ultimately a decrease in fruit yield.

[0003] In staple crops, a large number of studies have been conducted on fertility, especially on male sterility genes, of which dozens have been discovered. Male sterility is mainly caused by mutation or deletion of this gene, including transcriptional regulation, RNA metabolism, amino acid metabolism, signal transduction and pollen wall synthesis. Male sterility (MS) refers to the abnormal development of male reproductive organs during the sexual reproductive development stage, which results in the inability to produce pollen with normal functions, while the plant's vegetative and reproductive stages develop normally. Plants with male sterility phenotypes cannot produce pollen with normal functions, so they can be used as female parents in hybrid seed production, thereby reducing the cost of artificial emasculation and effectively ensuring the purity of hybrid seeds. This technology has no obvious side effects on the environment and plants. At present, the use of male sterility technology for crop hybrid breeding and seed production is an important way to improve the utilization rate of hybrid vigor and yield.

[0004] At present, genetic research on male sterility is also mainly focused on the above aspects. Research on cytochrome c (CYTc) protein is mainly concentrated in animals, and there are few in plants. CYTc is mainly located between mitochondrial complexes III and IV, responsible for the transfer of electron chains. When cells are stimulated, a large amount of CYTc will be transferred from mitochondria to the cytoplasm, causing a disorder of intracellular calcium ion balance, leading to the loss of mitochondrial membrane potential, aggravating the process of cell apoptosis, and thus causing programmed death of tissues and organs. In plants, direct research on plant fertility after changes in CYTc gene expression has not been reported. In sunflower, PET1-CMS male sterility is caused by premature programmed cell death of tapetum cells, which then causes CYTc to be released from mitochondria into the cytoplasm, resulting in damage to the outer mitochondrial membrane and decreased respiration (Janneke Balk, Christopher J. Leaver. The PET1-CMS mitochondrial mutation in sunflower is Associated with premature programmed cell death and Cytochrome c Release. 2001. Plant Cell. 13(8): 1803-1818). There are no reports on whether the expression of CYTc gene directly affects fertility. Summary of the invention

[0005] The purpose of the present invention is to provide a Castanopsis affine fertility gene IpCYTc and application thereof.

[0006] In order to achieve the above objectives, the present invention provides the following technical solutions:

[0007] The present invention provides a Castanopsis affine fertility gene IpCYTc, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The present invention also provides a protein encoded by the Castanopsis affine gene IpCYTc, wherein the protein is any one of the following:

[0009] (A1) the amino acid sequence is the protein shown in SEQ ID NO.2;

[0010] (A2) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0011] The present invention also provides a biological material, which is an expression box, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the Castanopsis castanopsis fertility gene IpCYTc.

[0012] The present invention also provides the use of the Castanopsis castanopsis fertility gene IpCYTc or the protein encoded by the Castanopsis castanopsis fertility gene IpCYTc or the biological material in regulating plant fertility.

[0013] Preferably, the regulating plant fertility is by promoting or increasing the expression of the Castanopsis castanopsis fertility gene IpCYTc to reduce the fertility of the plant, or by inhibiting or decreasing the expression of the Castanopsis castanopsis fertility gene IpCYTc to improve the fertility of the plant.

[0014] Preferably, the plant is Castanopsis or rice.

[0015] The present invention also provides a method for regulating plant fertility, comprising the following steps:

[0016] (1) using a rice anther-specific promoter to overexpress the Castanopsis affine gene IpCYTc to construct a plasmid containing the Castanopsis affine gene IpCYTc;

[0017] (2) transferring the plasmid obtained in step (1) into plants to obtain a transgenic plant sterile line;

[0018] The plant is Castanopsis or rice.

[0019] Preferably, the nucleotide sequence of the rice anther-specific promoter is shown in SEQ ID NO.3.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The application of IpCYTc and its encoded protein in fertility control has not been reported before. The present invention adopts the method of GWAS analysis, and first screens out IpCYTc from the strong sex-related sites of Castanopsis sylvestris, which has a potential sex regulation mechanism. The expression strength of IpCYTc determines the sex orientation. Therefore, the present invention adopts a specific promoter method to perform overexpression verification, that is, using a rice anther-specific promoter to overexpress IpCYTc, and genetically transforming it into rice, it can be detected that the fertility of rice anthers decreases, providing new materials for the application of male sterile lines of rice and Castanopsis sylvestris in hybrid seed production. It is generally reported that whether the gene is expressed or not determines the fertility; and the regulation of fertility by IpCYTc is different from the general gene function, and it is the strength of expression that determines the fertility. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Sex association analysis and candidate gene expression level analysis of Castanopsis chinensis;

[0023] Figure 2 Schematic diagram of constructing the anther-specific promoter vector of the IpCYTc gene in Castanopsis affine;

[0024] Figure 3 Results of GFP fluorescence microscopy of pollen from wild-type Zhonghua 11 (WT) and anther-specific overexpressing IpCYTc (Bar = 100 μm);

[0025] Figure 4 Quantitative fluorescence expression results of IpCYTc in pollen from wild-type Zhonghua 11 (WT) and anther-specific overexpressing IpCYTc;

[0026] Figure 5 Results of iodine staining analysis of pollen from wild-type Zhonghua 11 (WT) and anther-specific overexpressing IpCYTc (Bar = 100 μm). Detailed implementation mode

[0027] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be construed as limiting the protection scope of the present invention. The idesia polycarpa materials used in the following embodiments are idesia polycarpa growing in the Institute of Botany, Chinese Academy of Sciences, Beijing Xiangshan. The variety of wild-type rice is the common japonica rice variety Zhonghua 11 (ZH11).

[0028] Example 1 Discovery of the idesia polycarpa fertility gene IpCYTc

[0029] In order to analyze the idesia polycarpa fertility-related genes, the present invention selected male and female idesia polycarpa from the Institute of Botany, Chinese Academy of Sciences, Beijing Xiangshan, and conducted gender association analysis. It was found that there were two candidate genes in the gender association interval of chromosome 19, namely IpCYTc and IpTFL. Then, combined with the RNA-seq data of anthers and carpels of male and female flowers, it was analyzed that IpTFL in the sex determination interval was basically not expressed, but the expression of the IpCYTc gene between anthers and carpels was related to organ apoptosis (see Figure 1 ). In other words, in female plants, the carpels develop, the anthers undergo programmed cell death, and the expression level of IpCYTc in the carpels is lower than that in the anthers; in male plants, the carpels undergo programmed cell death, the anthers develop, and the expression level of IpCYTc in the carpels is higher than that in the anthers. According to the annotation, it was found that this gene encodes the cytochrome c protein in mitochondria. Since there is research in animals showing that high expression of the IpCYTc protein will cause programmed organ death (OwYP, GreenDR, Hao Z, et al. 2008. Cytochrome c: functions beyond respiration. Nat. Rev. Mol. Cell Biol. 9(7): 532-542.). Therefore, IpCYTc was taken as the subsequent gene related to flower organ development.

[0030] The coding sequence of the IpCYTc gene is shown in SEQ ID NO.1; the protein encoded by this gene is named IpCYTc protein, and the amino acid sequence is shown in SEQ ID NO.2.

[0031] SEQ ID NO.1

[0032] ATGGCTGGAGGAGGGGTGATCCACCAGCTTCTGAGGAGAAAACTTCGGTCACACTCTGGTGTCCCTTCAGTTATATCTTCCTTCACCTCAAAGAAAGTTCATGATGATGCTGGATCTGTGGGTATGAAGTCCCTAAGAGCATTTGCTTTATTTGGAGCTGGACTTTCAGGGTTTCTGAGTTTTGCATCAGTAGCATCTGCCGACGAGGCTGAACATGGGTTAGAGTGTCCAAGCTATCCTTGGCCACACGAAGGGATTCTAAGTTCTTATGACCATGCTTCGATTCGTCGTGGTCACCAGGTTTACCAGCAAGTATGTGCATCTTGCCACTCCATGTCCCTGATTTCATACCGTGATCTGGTGGGTGTTGCATATACAGAAGAGGAGACCAAGGCTATGGCGGCAGAGATTGAGGTGGTTGATGGACCGAACGATGAGGGTGAGATGTTTACACGTCCGGGTAAACTCAGTGACCGTTTTCCTCAGCCATATTCGAATGAACAAGCAGCTAGATTTGCTAATGGAGGAGCATATCCTCCAGATTTAAGTCTTATTACAAAAGTAATACCTACCTTCTTGTTTAGAATTTTTTTTGGAAGTTGTAATGGTCTATTGTAG。

[0033] SEQ ID NO.2

[0034] MAGGGVIHQLLRRKLRSHSGVPSVISSFTSKKVHDDAGSVGMKSLRAFALFGAGLSGFLSFASVASADEAEHGLECPSYPWPHEGILSSYDHASIRRGHQVYQQVCASCHSMSLISYRDLVGVAYTEEETKAMAAEIEVVDGPNDEGEMFTRPGKLSDRFPQPYSNEQAARFANGGAYPPDLSLITKVIPTFLFRIFFGSCNGLL*。

[0035] Example 2 Construction of the IpCYTc Anther-Specific Expression Vector of Idesia polycarpa

[0036] According to the IpCYTc nucleotide sequence SEQ ID NO.1 provided herein, primers CYTc_ORF_F / R were designed for amplification. Referring to the anther-specific promoter P12 sequence (SEQ ID NO.3) provided by Rao et al. (Rao GS, Deveshwar P, Sharma M. et al. 2018. Evolvement of transgenic male-sterility and fertility-restoration system in rice for production of hybrid varieties. Plant Mol. Biol. 96:35-51.), primers Anther_F / R were designed for amplification. According to the GFP sequence (SEQ ID NO.4) on the pCAMBIA2301 vector, primers GFP_F / R were designed for amplification.

[0037] The primers used for PCR amplification are shown in Table 1 below:

[0038] Table 1 Primers Used in the PCR Reaction

[0039]

[0040] The reaction system and PCR reaction program for the said PCR amplification are shown in Tables 2 - 3 below:

[0041] Table 2 PCR Reaction System

[0042]

[0043]

[0044] Centrifuge briefly to mix evenly. The PCR reaction uses a touch-down annealing program, and the annealing temperature is reduced by 0.5 °C for the first 10 cycles. The set program is shown in Table 3 below:

[0045] Table 3 PCR Amplification Program

[0046] Step Temperature (°C) Time Remarks Pre-denaturation 95 5 min Denaturation 95 30s Annealing 62 30s -0.5℃ Extension 72 30s 10 cycles Denaturation 95 30s Annealing 57 30s Extension 72 30s 24 cycles Extension 72 5 min Storage 4 ∞

[0047] Take 15 μL of the PCR product and detect it by 1% agarose gel electrophoresis. The target bands of the reaction products are all about 3400 bp. Cut and recover the gel using the ordinary agarose gel DNA recovery kit produced by Novoprotein Scientific Inc., and the method refers to the kit instruction manual. Finally, the specific fragment amplified by the primer pair consisting of primer Anther_F and primer IpCYTc_ORF_R is called the pAnther-GFP-IpCYTc sequence.

[0048] Use the restriction endonucleases HindⅢ and SalⅠ produced by NEB to perform double digestion on the pCAMBIA2301 vector simultaneously. After digestion, recover the large fragment to obtain the digested pCAMBIA2301 vector.

[0049] Use the one-step recombination kit produced by Novoprotein Scientific Inc. to recombine the above pAnther-GFP-IpCYTc sequence onto the digested pCAMBIA2301. The operation method follows the kit instruction manual. After the two are ligated, take 5 μL of the ligation product and transfer it into Escherichia coli DH5α, and culture it on solid LB medium containing kanamycin (Kana) for 12 h. Pick monoclonal colonies and place them in liquid LB containing Kana for culture. After the colony PCR identification is a positive clone, extract the plasmid and perform double digestion verification. For the plasmid with successful verification, send it to Tsingke Biological for sequencing. After the sequencing is correct, the successfully constructed IpCYTc vector pAnther::GFP-IpCYTc is obtained (see Figure 2 ).

[0050] After the sequencing is correct, transfer the obtained pAnther::GFP-IpCYTc plasmid into Agrobacterium tumefaciens EHA105 to carry out rice genetic transformation work. The rice genetic transformation is completed by Wuhan Boyuan Biotechnology Co., Ltd. to obtain the T0 generation transgenic lines of pAnther::GFP-IpCYTc under the background of rice ZH11, that is, the T0 generation of rice IpCYTc anther-specific overexpression.

[0051] The nucleotide shown in SEQ ID NO.3

[0052]

[0053] The nucleotide shown in SEQ ID NO.4:

[0054] ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACCCTGGTCACCACCCTGACCTACGGCGTGCAGTGCTTCTCCCGTTACCCTGATCATATGAAGCGGCACGACTTCTTCAAGAGCGCCATGCCTGAGGGATACGTGCAGGAGAGGACCATCTTCTTCAAGGACGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAGGGAGACACCCTCGTCAACAGGATCGAGCTTAAGGGAATCGATTTCAAGGAGGACGGAAACATCCTCGGCCACAAGTTGGAATACAACTACAACTCCCACAACGTATACATCATGGCCGACAAGCAAAAGAACGGCATCAAAGCCAACTTCAAGACCCGCCACAACATCGAAGACGGCGGCGTGCAACTCGCTGATCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAAGGCGCGCCAAGCTATCACAAGTTTGTACAAAAAAGC。

[0055] Identification of T0 generation plants of transgenic rice pAnther::GFP-IpCYTc in Example 3

[0056] Wild-type rice ZH11 and the T0 generation anthers of pAnther::GFP-IpCYTc were subjected to transcriptional and protein expression level detection. The anther RNA was extracted and reverse transcribed into cDNA for real-time fluorescence quantitative PCR detection, and the expression of GFP-tagged protein was detected under a fluorescence microscope. The results are as Figure 3 and Figure 4 shown. In the T0 generation materials of pAnther::GFP-IpCYTc (pAnther::GFP-IpCYTc 1# and pAnther::GFP-IpCYTc 2# respectively), the IpCYTc gene was highly expressed at both the transcriptional and protein levels compared to wild-type rice ZH11 (WT).

[0057] Example 4. Observation of pollen fertility by I2-KI staining

[0058] Take the florets on the rice spikelets, place them on a clean glass slide, use forceps to peel off the glumes, take out the anthers, and squeeze out the pollen grains; after removing the excess impurities, add 1 drop of I2-KI (2 g of iodine, 3 g of KI, dissolved in 100 mL of ddH2O); after staining for a few seconds, cover with a cover glass and observe the staining situation under a microscope. The pollen grains that can be deeply stained by the chromosome are fertile pollen, and those with very light staining are aborted pollen. Count the total number of fertile and sterile gametes in multiple fields of view. As Figure 5 shown, the pollen fertility of wild-type rice ZH11 (WT) was basically 100%, while the fertility of the specifically overexpressed IpCYTc materials (pAnther::GFP-IpCYTc 1# and pAnther::GFP-IpCYTc 2# respectively) decreased significantly. This result indicates that the expression intensity of IpCYTc is positively correlated with anther apoptosis.

[0059] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A fertility gene IpCYTc of Castanopsis chinensis, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A protein encoded by the Castanopsis affine gene IpCYTc of claim 1, characterized in that: The protein is any of the following: (A1) the amino acid sequence is the protein shown in SEQ ID NO.2; (A2) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

3. A biological material, characterized in that, The biological material is an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line comprising the Castanopsis castanopsis fertility gene IpCYTc described in claim 1.

4. Use of the Castanopsis affine gene IpCYTc according to claim 1 or the biological material according to claim 3 in regulating plant fertility; The regulating plant fertility is to reduce the fertility of the plant by promoting or increasing the expression of the Castanopsis affine fertility gene IpCYTc; The plant is rice.

5. A method for regulating plant fertility, characterized in that, The following steps are involved: (1) Using a rice anther-specific promoter to overexpress the Castanopsis castanopsis fertility gene IpCYTc described in claim 1, to construct a plasmid containing the Castanopsis castanopsis fertility gene IpCYTc; (2) transforming the plasmid obtained in step (1) into plants to obtain a transgenic plant sterile line; The plant is rice.

6. The method according to claim 5, wherein The nucleotide sequence of the rice anther-specific promoter is shown in SEQ ID NO.3.

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