Application of ECT2 and ECT4 genes in regulating poplar leaf size and morphology

Abstract

This invention provides an application of the ECT2 and ECT4 genes in regulating poplar leaf size and morphology. By regulating the ECT2 and ECT4 genes, the leaf area of ​​poplar can be significantly reduced, the leaf margins smoother, and the leaves more rounded. This method is efficient and precise, providing a new strategy for the genetic improvement of poplar, and is applicable to forestry breeding and ecological restoration.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant genetic engineering, and in particular to the application of ECT2 and ECT4 genes in regulating the size and morphology of poplar leaves. BACKGROUND

[0002] As an important economic forest and model plant, the size and morphology of poplar leaves directly affect photosynthetic efficiency, canopy structure and wood yield. Traditional breeding methods have limitations in regulating leaf traits, such as long cycle and low efficiency. This kind of deficiency is mainly due to the dependence on a large number of hybrid combinations and random selection, which lacks precision. Moreover, traditional methods are limited by the time-consuming nature of phenotypic observation. With the development of technology, modern technologies such as molecular biology techniques, genetic engineering and optimized cultivation management measures can partially overcome these deficiencies. SUMMARY

[0003] The present application provides a method for regulating the size and morphology of poplar leaves by double-knocking out ECT2 and ECT4 genes. The present application obtains a transgenic line ect2ect4 of double-knocking out ECT2 and ECT4 genes of poplar through genetic transformation, and finds that compared with wild type WT, the leaf of ect2ect4 poplar is smaller, the leaf margin is smooth, and the leaf is round, indicating that the ECT2 and ECT4 genes of poplar have a regulatory effect on the size and morphology of the leaf. The method provided by the present application can efficiently and accurately obtain transgenic poplar with changed leaf morphology and size, thereby providing an important basis for sustainable forestry resource development and having important value for forest cultivation and ecological restoration.

[0004] Specifically, the present application provides the following technical solutions:

[0005] The first aspect of the present application provides the application of ECT2 and ECT4 genes in regulating the size and morphology of poplar leaves, wherein the nucleotide sequence of the ECT2 gene is shown as SEQ ID NO: 1, and the nucleotide sequence of the ECT4 gene is shown as SEQ ID NO: 2.

[0006] According to the embodiments of the present application, compared with wild type plants, the leaves of plants with deletion of ECT2 and ECT4 genes or with significantly reduced expression of ECT2 and ECT4 proteins are smaller and rounder.

[0007] According to the embodiments of the present application, the knockout of the ECT2 and ECT4 genes is realized by at least one of the following methods:

[0008] Crispr / Cas9, zinc finger nuclease, RNA interference, transcription activator-like effector nuclease.

[0009] According to an embodiment of the present application, the knockout of the ECT2 and ECT4 genes is achieved by Crispr / Cas9 technology.

[0010] The second aspect of the present application provides an application of ECT2 protein and ECT4 protein in regulating the size and morphology of poplar leaves, wherein the amino acid sequence of the ECT2 protein is shown as SEQ ID NO: 3, and the amino acid sequence of the ECT4 protein is shown as SEQ ID NO: 4.

[0011] The third aspect of the present application provides a method for producing a transgenic poplar, comprising:

[0012] knocking out the ECT2 and ECT4 genes in a wild-type poplar to obtain a transgenic poplar;

[0013] wherein the nucleotide sequence of the ECT2 is shown as SEQ ID NO. 1, and the nucleotide sequence of the ECT4 is shown as SEQ ID NO. 2.

[0014] According to an embodiment of the present application, the expression amount of the ECT2 and ECT4 proteins in the transgenic poplar is significantly reduced compared with that in a wild-type poplar.

[0015] wherein the amino acid sequence of the ECT2 protein is shown as SEQ ID NO: 2, and the amino acid sequence of the ECT4 protein is shown as SEQ ID NO: 4.

[0016] According to an embodiment of the present application, the knockout of the ECT2 and ECT4 genes is achieved by at least one of the following methods:

[0017] Crispr / Cas9, zinc finger nuclease, RNA interference, transcription activator-like effector nuclease.

[0018] According to an embodiment of the present application, the leaves of the transgenic poplar are significantly smaller and rounder than the leaves of a wild-type poplar.

[0019] The fourth aspect of the present application provides a method for regulating the size and morphology of poplar leaves, comprising:

[0020] regulating the expression amount of the ECT2 and ECT4 genes in a poplar, so that the expression amount of the ECT2 and ECT4 proteins in the poplar is significantly reduced compared with that in a wild-type poplar.

[0021] The present application has at least the following beneficial effects:

[0022] By designing specific sgRNA, constructing CRISPR / Cas9 vector, and obtaining transgenic poplar with knockout of ECT2 and ECT4 genes through Agrobacterium-mediated transformation, a new strategy for genetic improvement of poplar is provided.

[0023] In the project, 84K poplar is used as background material, and two ect2ect4 double knockout lines L2 and L3 are obtained by CRISPR / Cas9 technology and Agrobacterium-mediated genetic transformation of poplar. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Figure 1 is a leaf phenotype diagram of wild type and mutant lines ect2ect4 according to an embodiment of the present application (WT refers to wild type, L2 and L3 refer to two different lines of ect2ect4).

[0025] Figure 2 Figure 2 is a leaf area statistical diagram of wild type and mutant lines ect2ect4 according to an embodiment of the present application (WT refers to wild type, L2 and L3 refer to two different lines of ect2ect4). DETAILED DESCRIPTION

[0026] The embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings, and the embodiments described below by referring to the accompanying drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0027] Currently, there are few reports on the simultaneous knockout of poplar ECT2 and ECT4 genes and its application in leaf size and morphology regulation. ECT2 and ECT4 genes play an important role in the growth and development of poplar. In the present application, ECT2 and ECT4 genes are specifically knocked out by CRISPR / Cas9 technology, and it is found that the leaf size and morphology of poplar can be efficiently regulated, which provides a new strategy for genetic improvement of poplar.

[0028] The ECT2 gene mentioned in the present application has the nucleotide sequence shown in SEQ ID NO: 1.

[0029] The nucleotide sequence shown in SEQ ID NO: 1 is as follows:

[0030]

[0031] The ECT4 gene mentioned in the present application has the nucleotide sequence shown in SEQ ID NO: 2.

[0032] The nucleotide sequence shown in SEQ ID NO: 2 is as follows:

[0033]

[0034] The ECT2 gene coding protein in the present application is ECT2 protein, and its amino acid sequence is shown as SEQ ID NO: 3:

[0035] MPEPTSKIQPSDRSVTPVLSNFMDPTVCYLPNGYQSYYYGGYNGAGEWDDYSKYLNPEGVDMVSGVYGDNGSAMYPHGYWYGPYSPYSPAASPVPTMGNDGQLYGPQHYQYPPPYFQPLTPSGEPFTPSHVAPSQGDLSISTATDQKPLPVETAKENSNGIANGVDVKGSNGAVPYKPKYQNSYGRGGFTKGLPASGYKDLKSRFDRLQPDSPLLDTSVLSSGLYKNTEISSSFSKAGNAPSSRNQNFHQNSHFMGWQHPALAPGVGSTHGYMNRMYPNKFYGQYGNGFKSGMGFGSGGYNAGINGHGWLAIDSKYKPKGRGNGYFGYHNDSIDGLNELNRGPRAKGYFKNQKGFVPATVAVKGQSVPSSDTNVEEKDKTTVVPDREQYNKADFPEEYDNAKFFIIKSYSEDDVHKCIKYNVWASTPNGNKKLDAAYQEAEQKSGGCPVFLLFSVNTSGQFVGLAEMTGRVDFDKSVEYWQQDKWTGYFPVKWHIVKDVPNSFLKHITLENNENKPVTNSRDTQEVKLEQGLKLIKIFKDHSSKTCILDDFVFYEDREKMIQEKKAKQQQLKKQVWDGKPCEEKKEIANGS (SEQ ID NO: 3)

[0036] The ECT4 gene coding protein in the present application is ECT4 protein, and its amino acid sequence is shown as SEQ ID NO: 4:

[0037] MATLSSSAEQTSDLLQKLSIDSQTKTLDMPEPTNKIQPSDRSVTPVLSNFMDPTVCYLPNGY PSYYYGGYNGTGEWDDYSKYLNTEGVDMTSGNGSAMYPHGYWYGPYGPYSPAASPVPTMG NDGQLYGPQHYQYPTPYFQPLTPTSEPFIPSHVAPSQGDLSITTAADQKSLPVETGKENSNG IANGGDVKGANGAVPYKPKYQNSYGRGSYTKGIPASGYKDLRSCFDRLQPDIPLLDSSVLSD GLYRNTDISSSFSKASNAPSSRNQNFHQNSHFMGWQHPGLASGMGSTHGYMNRMYSNKLY GQYGNGFKSGVGFGSGGYNAGINGQGWLPIDSKYKPKGQGNGYFGFRNENIDGLNELNRGPR AKGYFKNQKGFVPSTVAVKGQSVPSSDANAEEKDKTTEVPDREQYNKADFPVEYVDAKFFI IKSYSEDDVHKCIKYNVWASTPNGNKKLDAAYQEAGQKSGGCPVFLLFSVNTSGQFVGLAE MTGRVDFDKSVEYWQQDKWTGYFPVKWHFVKDVPNSLLKHITLENNENKPVTNSRDTQEVKL EQGLEMIKIFKEHSSKTCILDDFGFYEDREKMIQEKKAKQQQLKKQVWDGKPSDEKKELVN GS (SEQ ID NO: 4)

[0038] The sgRNA molecule-containing sequence described in the present application for the ECT2 and ECT4 genes is shown as SEQ ID NO: 5:

[0039] ACCCTGACACTGGAATCGGCAGCAAAGGATTTACTTTAAATTTTTTCTTATGCAGCCTGTGATGGATAACTGAATCAAACAAATGGCGTCTGGGTTTAAGAAGATCTGTTTTGGCTATGTTGGACGAAACAAGTGAACTTTTAGGATCAACTTCAGTTTATATATGGAGCTTATATCGAGCAATAAGATAAGTGGGCTTTTTATGTAATTTAATGGGCTATCGTCCATAGATTCACTAATACCCATGCCCAGTACCCATGTATGCGTTTCATATAAGCTCCTAATTTCTCCCACATCGCTCAAATCTAAACAAATCTTGTTGTATATATAACACTGAGGGAGCAACATTGGTCACACAGTTGGATCCATGAAGTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGACCAAGACACTGGAATCGGCAGCAAAGGAATAAGCTTATGATTTCTTTTTTCTTACGAATTTTGCGTCCCACATCGGTAAGCGAGTGAAGAAATAACTGCTTTATAtATGGCTACAAAGCACCATTGGTCACTGTGTGCTATCTTCCTAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTT (SEQ ID NO: 5)

[0040] The application also provides a method for regulating the size and shape of poplar leaves, comprising: knocking out the ECT2 and ECT4 genes to obtain a mutant strain. The mutant strain has significantly smaller and rounder leaves than the wild-type poplar (according to the specific embodiments, the size of the leaves of the poplar after gene knockout is 40% to 70% of the size of the leaves of the poplar without knockout of the ECT2 and ECT4 genes, for example, 40% to 60%). According to the specific embodiments, the leaf edges of the mutant strain are smooth.

[0041] The available methods include, but are not limited to, Crispr / Cas9, zinc finger nuclease, RNA interference, transcription activator-like effector nuclease, etc. The CRISPR / Cas9 system is modified from the adaptive immune system of bacteria, comprising a Cas9 nuclease and a single-stranded guide RNA (sgRNA). The sgRNA recognizes the target DNA sequence through base pairing, and the Cas9 cuts the DNA double strand near the PAM sequence (usually NGG), inducing double-strand breaks (DSB); this method can achieve efficient and accurate targeting. Zinc finger nuclease (ZFN) is fused from zinc finger protein (ZFP) DNA binding domain and FokI nuclease. Each zinc finger module recognizes a 3bp DNA sequence, and multiple modules are concatenated to achieve specific binding. FokI needs to form a dimer to cut DNA, so it needs to be used in pairs. RNA interference (RNAi) uses double-stranded RNA molecules such as small interfering RNA (siRNA) or microRNA (miRNA) to degrade target mRNA or inhibit its translation through RNA-induced silencing complex (RISC), thereby down-regulating gene expression. Transcription activator-like effector nuclease (TALEN) is fused from TALE protein DNA binding domain and FokI nuclease. Each TALE repeat unit recognizes a specific base (such as NI=A, HD=C), and targets DNA by combining repeat sequences. Similar to ZFN, it needs to be used in pairs to activate FokI cutting. According to a preferred embodiment, the method used is the Crispr-Cas9 method, which can achieve ECT2 and ECT4 gene knockout. Target sites are designed for ECT2 and ECT4 genes, and then connected to an expression vector containing Cas9. The available expression vectors include, but are not limited to, PMDC164, pYLCRISPR / Cas9, pHSbdcas9i-Cr-PDS, etc.

[0042] In this document, "significant" means statistically significant difference, and without special instructions, it means p value < 0.05.

[0043] The present application provides a transgenic plant, wherein the ECT2 and ECT4 genes are double knockout (crispr ect2ect4).

[0044] The technical solutions of the present application are described below through specific examples, and it should be noted that these examples are only used to facilitate the understanding of those skilled in the art, and should not be regarded as a limitation on the scope of protection of the present application. Unless otherwise specified, the reagents used in the examples can be obtained by commercial purchase.

[0045] Example 1 Design of Targeting Sequence and Vector Construction

[0046] Target sites were designed for ECT2 and ECT4 genes through online database http: / / skl.scau.edu.cn / targetdesign / , and then were connected to the expression vector containing Cas9 (the sequence constructed is shown as SEQ ID NO: 5). The target site sequence used is as follows:

[0047] Target1 PAM (target site 1): CCAACTTCATGGATCCAACTGTG (SEQ ID NO: 6)

[0048] Target2 PAM (target site 2): ACTGTGTGCTATCTTCCTAATGG (SEQ ID NO: 7)

[0049] The sgRNA sequence used is as follows:

[0050] GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGG (SEQ ID NO: 8).

[0051] Example 2 Poplar genetic transformation and screening

[0052] Example 2 transgenic poplar was obtained by the following method:

[0053] (1) 84K poplar was used as material to prepare callus.

[0054] (2) The double knockout expression vector constructed in Example 1 was introduced into poplar callus by Agrobacterium (strain GV3101) mediated transformation.

[0055] (3) Transgenic callus was screened on medium containing hygromycin, and transgenic plants were induced to differentiate.

[0056] The components in the medium are respectively: MS powder 2.215 g / L, sucrose 30 g / L, agar 5 g / L, NAA 0.02 mg / L, IBA 0.05 mg / L, hygromycin 1.5 mg / L.

[0057] (4) Genomic DNA extraction was performed on the transgenic plants, PCR amplification of poplar ECT2 and ECT4 genes and their target regions was performed, and Sanger sequencing was performed.

[0058] The primers used are as follows:

[0059] ECT2 gene:

[0060] F1: GGAGTGAGTACGGTGTGCCAGCAAGGTAATTTTCACCCTTAAAG (SEQ ID NO:9)

[0061] R1: GAGTTGGATGCTGGATGGCTCACCTCCATAGTAATAGGATTG (SEQ ID NO:10)

[0062] ECT4 gene:

[0063] F2: GGATGAGTACGGTGTGCGGTTTAGGCTTGTGCTTATATTTG (SEQ ID NO: 11)

[0064] R2:GAGTTGGATGCTGGATGGCTTCAGCATGTTAAACTCACC (SEQ ID NO:12)

[0065] The PCR amplification system and procedure are shown below:

[0066] Table 1: PCR amplification system

[0067]

[0068] Table 2: PCR amplification program

[0069]

[0070] Sanger sequencing results showed that the ECT2 and ECT4 genes were successfully knocked out.

[0071] Example 3: Phenotypic Analysis of Transgenic Poplar

[0072] Tissue culture seedlings grown in sterile tissue culture bottles for 4 weeks were hardened off and then transplanted to a greenhouse for further growth. After 3 months of growth, the phenotypic characteristics and development of their leaves were observed.

[0073] The results showed that, compared to wild-type (WT) poplar, the leaves of poplar crispr ect2ect4 were significantly smaller (e.g., Figure 1 As shown, Figure 1 L2 and L3 represent two transgenic poplar lines, and their leaf edges are smoother.

[0074] By statistically analyzing the leaf area of ​​WT and crisp2ect4, it was found that (e.g. Figure 2 As shown in the figure, the leaf area of ​​crisper ect2ect4 was significantly reduced.

[0075] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and that changes, modifications, substitutions and variations can be made by those skilled in the art without departing from the scope of the present application.

Claims

1. A method of producing a transgenic poplar tree, characterized by, Comprise: knocking out ECT2 and ECT4 genes in wild type poplar to produce a transgenic poplar; wherein the nucleotide sequence of the ECT2 is shown as SEQ ID NO. 1; and the nucleotide sequence of the ECT4 is shown as SEQ ID NO.

2.

2. The method of claim 1, wherein, The expression amount of ECT2 and ECT4 proteins in the transgenic poplar is significantly lower than that in wild type poplar. wherein the amino acid sequence of the ECT2 protein is shown as SEQ ID NO: 2; and the amino acid sequence of the ECT4 protein is shown as SEQ ID NO:

4.

3. The method of claim 1, wherein, The knockout of the ECT2 and ECT4 genes is achieved by at least one of the following methods: Crispr / Cas9, zinc finger nuclease, RNA interference, transcription activator-like effector nuclease.

4. The method of claim 1, wherein, The leaves of the transgenic poplar are significantly smaller and rounder than those of wild type poplar.

5. A method of modulating leaf size and morphology in Populus comprising, Comprise: regulating the expression amount of ECT2 and ECT4 genes in poplar, so that the expression amount of ECT2 and ECT4 proteins in the poplar is significantly lower than that in wild type poplar; the nucleotide sequence of the ECT2 gene is shown as SEQ ID NO: 1; and the nucleotide sequence of the ECT4 gene is shown as SEQ ID NO: 2.

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