Idesia polycarpa fertility gene IpCYTc and application thereof

Abstract

The invention relates to the technical field of gene engineering, in particular to an idesia fertility gene IpCYTc and application thereof, and the nucleotide sequence of the idesia fertility gene IpCYTc is shown as SEQ ID NO.1. According to the idesia fertility gene IpCYTc, IpCYTc is overexpressed by utilizing a rice anther specific promoter and is genetically transformed into rice, the rice anther fertility reduction can be detected, and the idesia fertility gene IpCYTc can be used for detecting the rice anther fertility reduction. A new material is provided for the application of the male sterile line of the rice and the idesia polycarpa 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 The results of GFP fluorescence microscopy of wild-type Zhonghua11 (WT) and anther-specific overexpression of IpCYTc pollen (Bar=100 μm);

[0025] Figure 4 The figure shows the fluorescence quantitative expression results of IpCYTc in the pollen of wild-type Zhonghua 11 (WT) and anther-specific overexpression of IpCYTc;

[0026] Figure 5 The results of pollen iodine staining analysis of wild-type Zhonghua11 (WT) and anther-specific overexpression of IpCYTc are shown (Bar=100 μm). DETAILED DESCRIPTION

[0027] The technical scheme provided by the present invention is described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present invention. The tung oil tree material used in the following embodiments is the tung oil tree grown in the Institute of Botany, Chinese Academy of Sciences, Xiangshan, Beijing. The variety of wild rice is the commonly used japonica rice variety Zhonghua 11 (ZH11).

[0028] Example 1 Discovery of the Fertility Gene IpCYTc in Castanopsis chinensis

[0029] In order to analyze the fertility-related genes of Castanopsis chinensis, the present invention selected male and female Castanopsis chinensis plants from the Institute of Botany, Chinese Academy of Sciences, Beijing, and conducted sex association analysis. It was found that there were two candidate genes, IpCYTc and IpTFL, in the sex association interval of chromosome 19. Then, combined with the RNA-seq data of anthers and carpels of male and female flowers, the analysis found 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 carpel develops and the anther undergoes programmed death, and the expression level of IpCYTc in the carpel is lower than that in the anther; in male plants, the carpel undergoes programmed death and the anther develops, and the expression level of IpCYTc in the carpel is higher than that in the anther. According to the annotation, it was found that this gene encodes cytochrome c protein in mitochondria. Studies in animals have shown that high expression of IpCYTc protein can 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 used as a subsequent gene related to floral 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 anther-specific expression vector of IpCYTc in Castanopsis affine

[0036] According to the IpCYTc nucleotide sequence SEQ ID NO.1 provided herein, the primer CYTc_ORF_F / R was designed for amplification. With reference 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.), the primer Anther_F / R was designed for amplification. According to the GFP sequence (SEQ ID NO.4) on the pCAMBIA2301 vector, the primer GFP_F / R was designed for amplification.

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

[0038] Table 1 Primers used in PCR reaction

[0039]

[0040] The reaction system and PCR reaction procedure of the 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 in the first 10 cycles. The program settings are shown in Table 3 below:

[0045] Table 3 PCR amplification program

[0046] step Temperature(℃) time Remark Pre-denaturation 95 5min transsexual 95 30s annealing 62 30s -0.5℃ extend 72 30s 10cycles transsexual 95 30s annealing 57 30s extend 72 30s 24cycles extend 72 5min save 4 ∞

[0047] 15 μL of PCR product was taken and detected by 1% agarose gel electrophoresis. The target bands of the reaction products were all about 3400 bp. The gel was cut and recovered using a common agarose gel DNA recovery kit produced by Novozymes. The method was referred to the kit instructions. Finally, the specific fragment amplified by the primer pair consisting of primer Anther_F and primer IpCYTc_ORF_R was called pAnther-GFP-IpCYTc sequence.

[0048] The pCAMBIA2301 vector was double-digested with restriction endonucleases HindⅢ and SalⅠ produced by NEB Company, and the large fragment was recovered after digestion to obtain the digested pCAMBIA2301 vector.

[0049] The pAnther-GFP-IpCYTc sequence was recombined into the pCAMBIA2301 after restriction digestion using a one-step recombination kit produced by Novozymes. The operation method was in accordance with the instructions of the kit. After the two were connected, 5 μL of the connection product was transferred into Escherichia coli DH5α and cultured on solid LB medium containing kanamycin (Kana) for 12 h. A single clone colony was picked and placed in liquid LB containing Kana for culture. After the bacterial liquid PCR was identified as a positive clone, the plasmid was extracted and double restriction digestion was performed. The successfully verified plasmid was sent to Qingke Bio for sequencing. After the sequencing was correct, the successfully constructed IpCYTc vector pAnther::GFP-IpCYTc (see Figure 2 ).

[0050] After sequencing was correct, the pAnther::GFP-IpCYTc plasmid was transferred into Agrobacterium EHA105 for rice genetic transformation. The rice genetic transformation was completed by Wuhan Boyuan Biotechnology Co., Ltd., and the T0 generation transgenic strain of pAnther::GFP-IpCYTc in the rice ZH11 background was obtained, that is, the T0 generation of rice IpCYTc anther-specific overexpression.

[0051] 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 pAnther::GFP-IpCYTc T0 generation anthers were used for transcriptional and protein expression testing. Anther RNA was extracted and reverse transcribed into cDNA for real-time fluorescence quantitative PCR testing. GFP-tagged protein expression was detected under a fluorescence microscope. The results are shown in Figure 2. Figure 3 and Figure 4 As shown, the IpCYTc gene in the pAnther::GFP-IpCYTc T0 generation materials (pAnther::GFP-IpCYTc 1# and pAnther::GFP-IpCYTc 2#, respectively) was highly expressed at both the transcriptional and protein levels in the wild-type rice ZH11 (WT).

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

[0058] Take the floret from the rice spikelet, place it on a clean glass slide, peel the husk with tweezers, take out the anther, and squeeze out the pollen grains; remove the excess impurities and add 1 drop of I2-KI (2g of single substance, 3g of KI, dissolved in 100mL ddH2O); after staining for a few seconds, add a coverslip and observe the staining under a microscope. The pollen that can be darkly stained by chromosomes is fertile pollen, and the pollen that is very lightly stained is aborted pollen. Count the total number of fertile and sterile gametes in multiple fields of view. Figure 5 As shown, the pollen grain breeding 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) was significantly reduced. This result indicates that the expression intensity of IpCYTc is positively correlated with anther apoptosis.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection 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 biomaterial, 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 castanopsis fertility gene IpCYTc according to claim 1, or the protein encoded by the Castanopsis castanopsis fertility gene IpCYTc according to claim 2, or the biological material according to claim 3 in regulating plant fertility.

5. The use according to claim 4, characterized in that: The regulation of 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.

6. The use according to claim 4 or 5, characterized in that: The plant is Castanopsis or rice.

7. 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) transferring the plasmid obtained in step (1) into plants to obtain a transgenic plant sterile line; The plant is Castanopsis or rice.

8. The method according to claim 7, characterized in that The nucleotide sequence of the rice anther-specific promoter is shown in SEQ ID NO.3.

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