Application and method of AtIDD7 gene in regulation and control of plant growth and development

Abstract

The invention relates to the technical field of biology, in particular to application and a method of an AtIDD7 gene in regulation and control of plant growth and development. The application of the plant AtIDD7 protein in regulation and control of plant growth and development is identified for the first time by researching the plant AtIDD7 protein. By overexpressing the AtIDD7 protein in the plant, the growth and development of overground and underground parts of the plant can be regulated and controlled, and an excellent gene resource is provided for the improvement of the growth and development of the plant.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an application and method of AtIDD7 gene in regulating plant growth and development. Background Art

[0002] The proteins encoded by IDD (INDETERMINATEDOMAIN) family genes are conserved transcription factors in plants. They were first defined by the ID domain of the flowering transition gene INDETERMINATE1 (ID1) in maize. The encoded proteins have two typical (C2H2) and two atypical (C2HC) zinc finger structures (Colasanti et al. 1998; Englbrecht et al. 2004; Colasanti et al. 2006). Amino acid sequence alignment results show that the four zinc finger structures are conserved in many plants such as Arabidopsis, rice and maize (Kumar et al. 2019; Colasanti et al. 2006).

[0003] Arabidopsis IDD family genes are involved in multiple processes such as plant development, sugar homeostasis, gravity response, and plant hormone regulation (Kumar et al. 2019; Coelho et al. 2018). IDD1 / ENY regulates seed development by affecting ABA balance through GA (Feurtado et al., 2011); IDD2 / GAF1 regulates seed germination process in a GA-dependent manner (Fukazawa et al., 2014; Feurtado et al., 2011); AtIDD3 / MGP, AtIDD4 / IME, AtIDD6 / BLJ, AtIDD8 / NUTCRACKER (NUC), AtIDD9, and AtIDD10 / JACKDOW (JKD) are known to be involved in Arabidopsis root development (Welch et al., 2007; Long et al., 2015; Moreno-Risueno et al., 2015; Ogasawara et al., 2011). AtIDD14, AtIDD15 and AtIDD16 regulate the morphological construction and gravity response of aerial organs by promoting the synthesis and transport of auxin (Cui et al. 2013). Another study showed that AtIDD15 regulates the gravity response of Arabidopsis inflorescence stems under high temperature by producing different spliceosomes (Kim et al. 2016). The functions of IDD7, IDD11, IDD12 and IDD13 genes in this family have not been reported.

[0004] Given the importance of this family of genes in plant growth and development, it is necessary to study the functions of other IDD genes. Summary of the invention

[0005] In order to solve the above problems, the present invention provides the application and method of AtIDD7 gene in regulating plant growth and development. The present invention can regulate the growth and development of the above-ground and underground parts of plants by overexpressing AtIDD7 gene in plants, thereby providing excellent gene resources for improving plant growth and development.

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

[0007] The invention provides application of AtIDD7 gene in regulating plant growth and development.

[0008] Preferably, overexpression of the AtIDD7 gene negatively regulates plant growth and development.

[0009] Preferably, overexpression of the AtIDD7 gene inhibits plant leaf development.

[0010] Preferably, overexpression of the AtIDD7 gene inhibits the elongation of the plant's main root.

[0011] Preferably, the plant comprises Arabidopsis thaliana.

[0012] Preferably, the nucleotide sequence of the AtIDD7 gene is shown as SEQ ID No.1.

[0013] Preferably, the amino acid sequence of the protein encoded by the AtIDD7 gene is shown as SEQ ID No.2.

[0014] The present invention also provides a method for regulating plant growth and development, comprising the following steps:

[0015] 1) constructing the AtIDD7 gene in the application described in the above technical solution into an expression vector containing EGFP or SRDX to obtain a recombinant expression vector;

[0016] 2) transforming the recombinant expression vector obtained in step 1) into Agrobacterium tumefaciens to obtain transformed bacteria;

[0017] 3) Infecting plants with the transformed bacteria obtained in step 2) to regulate plant growth and development.

[0018] Preferably, the expression vector in step 1) includes pAMBIA1302-35S-EGFP or pCXSN-35S-HA-SRDX.

[0019] Preferably, the Agrobacterium tumefaciens in step 2) is Agrobacterium tumefaciens GV3103;

[0020] The plants in step 3) include Arabidopsis thaliana, and Arabidopsis thaliana flowers are infected.

[0021] Beneficial effects of the present invention:

[0022] The present invention identifies for the first time the application of plant AtIDD7 protein in regulating plant growth and development through research on AtIDD7 protein in plants.

[0023] The present invention can regulate the growth and development of the above-ground and underground parts of plants by overexpressing the AtIDD7 protein in plants, thereby providing an excellent gene resource for improving the growth and development of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0025] Figure 1 , a is the phenotype of 35S:EGFP and 35S:EIDD7-GFP T2 generation materials grown on 1 / 2MS medium to the 8th day; b is the protein localization of EIDD7-GFP in the main root of Arabidopsis thaliana of 35S:EIDD7-GFP transgenic material; c is the phenotype of 35S:SRDX and 35S:IDD7-SRDXT2 generation materials grown on 1 / 2MS medium to the 8th day; d is the main root length statistics of 35S:EGFP and 35S:EIDD7-GFP T2 generation materials grown on 1 / 2MS medium to the 8th day; e is the main root length statistics of 35S:SRDX and 35S:IDD7-SRDXT2 generation materials grown on 1 / 2MS medium to the 8th day. DETAILED DESCRIPTION

[0026]

[0027] In the present invention, the amino acid sequence of the protein encoded by the AtIDD7 gene is shown in SEQ ID No. 2, which is as follows:

[0028] MMMNRDILFHQQQQQQMEENMSNLTSASGDQASVSSGNRTETSGSNINQHHQEQCFVPQSSLKRKRNQPGNPDPEAEVMALSPKTLMATNRFICEVCNKGFQRDQNLQLHKRGH NLPWKLKQRSNKDVVRKKVYVCPEPGCVHHHPSRALGDLTGIKKHFFRKHGEKKWKCEKCSKKYAVQSDWKAHAKTCGTKEYKCDCGTLFSRRDSFITHRAFCDALAEESARAM PNPIMIQASNSPHHHHHQTQQNIGFSSSSQNIISNSNLHGPMKQEESQHHYQNIPPWLISSNPNPNGNNGNLFPPVASSVNTGRSSFPHPSPAMSATALLQKAAQMGSTKSTTP EEEERSSRSSYNNLITTTMAAMMTSPPEPGFQDYYMMNHQHHGGGEAFNGGFVPGEEKNDVVDDGGGETRDFLGLRSLMSHNEILSFANNLGNCLNTSATEQQQQQHSHQD.

[0029] In the present invention, overexpression of AtIDD7 gene preferably negatively regulates plant growth and development. In the present invention, overexpression of AtIDD7 gene preferably inhibits plant leaf development. In the present invention, overexpression of AtIDD7 gene preferably inhibits plant taproot elongation. In the present invention, the plant preferably includes Arabidopsis thaliana.

[0030] The present invention also provides a method for regulating plant growth and development, comprising the following steps:

[0031] 1) constructing the AtIDD7 gene in the application described in the above technical solution into an expression vector containing EGFP or SRDX to obtain a recombinant expression vector;

[0032] 2) transforming the recombinant expression vector obtained in step 1) into Agrobacterium tumefaciens to obtain transformed bacteria;

[0033] 3) Infecting plants with the transformed bacteria obtained in step 2) to regulate plant growth and development.

[0034] The present invention constructs the AtIDD7 gene in the application described in the above technical solution onto an expression vector containing EGFP or SRDX to obtain a recombinant expression vector. In the present invention, the expression vector preferably includes pAMBIA1302-35S-EGFP or pCXSN-35S-HA-SRDX. The present invention does not specifically limit the method for constructing the AtIDD7 gene onto an expression vector containing EGFP or SRDX, and a conventional method for constructing a gene onto a vector can be used.

[0035] The present invention transforms the obtained recombinant expression vector into Agrobacterium tumefaciens to obtain a transformed bacterium. In the present invention, the Agrobacterium tumefaciens is preferably Agrobacterium tumefaciens GV3103. The present invention has no particular limitation on the method for transforming the recombinant expression vector into Agrobacterium tumefaciens, and a conventional method can be used.

[0036] The present invention uses the obtained transformed bacteria to infect plants to achieve the regulation of plant growth and development. In the present invention, the plant preferably includes Arabidopsis thaliana, and Arabidopsis thaliana flowers are infected. The present invention does not specifically limit the method of infecting plants with the transformed bacteria, and those skilled in the art can use conventional methods.

[0037] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1

[0039] Application and regulation method of AtIDD7 gene in regulating plant growth and development:

[0040] (I) Construction of 35S:EIDD7-GFP and 35S:IDD7-SRDX transgenic plants

[0041] 1. Construction of 35S:EIDD7-GFP and 35S:IDD7-SRDX recombinant vectors

[0042] In order to obtain plants overexpressing the IDD7 gene, the expression vectors 35S:EIDD7-GFP and 35S:IDD7-SRDX of the wild-type IDD7 gene driven by the CaMV 35S (Cauliflower mosaicvirus 35S) promoter were used for plant transformation.

[0043] The CDS (without the stop codon) sequence of IDD7 was obtained from the Tair (https: / / www.arabidopsis.org / ) website by gene number, and the BglII restriction site of pCAMBIA1302-35S-EGFP and pCXSN-35S-HA-SRDX) was selected. The primers were designed using the In-Fusion online primer design tool on the TaKaRa website:

[0044] EIDD7-GFP-F (SEQ ID No. 3):

[0045] 5'-CGAGCTGTACAGATCTATGATGATGAACAGAGACATATT-3';

[0046] EIDD7-GFP-R (SEQ ID No. 4):

[0047] 5'-GGCCGCTTTAAGATCTATCTTGGTGGCTATGTTGTTGT-3';

[0048] IDD7-SRDX-F (SEQ ID No. 5):

[0049] 5'-AACTAGTGGAAGATCTATGATGATGAACAGAGACATATT-3');

[0050] IDD7-SRDX-R (SEQ ID No. 6):

[0051] 5'-AATCCAAATCAGATCTATCTTGGTGGCTATGTTGTTGT-3'.

[0052] The CDS sequence was amplified using Col wild-type cDNA as a template to obtain a 1365bp PCR product. The pAMBIA1302-35S-GFP and pCXSN-35S-HA-SRDX plasmids were digested with BglII single enzyme digestion method, and the CDS sequences were seamlessly connected to the target vectors by the In-Fusion method. After sequencing, the IDD7 gene sequence shown in Sequence 2 was also obtained to obtain pAMBIA1302-35S-IDD7-GFP and pCXSN-35S-IDD7-SRDX plasmids, proving that the vector construction was correct.

[0053] Both pAMBIA1302-35S-GFP and pCXSN-35S-HA-SRDX vectors carry a hygromycin resistance gene.

[0054] 2. Obtaining IDD7 gene transgenic Arabidopsis

[0055] (1) The recombinant plasmids pAMBIA1302-35S-IDD7-GFP and pCXSN-35S-IDD7-SRDX were respectively introduced into the Agrobacterium strain GV3101 to obtain recombinant Agrobacterium GV3101 / pAMBIA1302-35S-IDD7-GFP and GV3101 / pCXSN-35S-IDD7-SRDX;

[0056] The method is to add the recombinant plasmid to 100μl Agrobacterium competent medium, put it on ice for 5min, freeze it in liquid nitrogen for 20s, and then water bath it at 37℃ for 5min. Add 700μl LB liquid medium without antibiotics, and culture it at 28℃ and 200rpm for 3h. Centrifuge at 3000rpm to remove the supernatant and keep 100μl LB to resuspend the bacteria, spread it on a plate of resistant LB (containing 50ug / ml kanamycin), and culture it upside down at 28℃. At the same time, the empty vector plasmids pAMBIA1302-35S-GFP and pCXSN-35S-SRDX without the IDD7 gene were subjected to the same operation as above, and used as the control group later.

[0057] (2) The recombinant plasmids pAMBIA1302-35S-EIDD7-GFP and pCXSN-35S-IDD7-SRDX were respectively introduced into wild-type Arabidopsis Col-0 to obtain T1 generation IDD7-transfected Arabidopsis, and the specific method was as follows:

[0058] A single clone was selected and placed in 2 ml of liquid LB medium containing kanamycin and rifampicin resistance, and cultured at 28°C, 250 rpm for 16 hours; 0.2 ml of the bacterial solution was added to 100 ml of liquid LB medium containing kanamycin and rifampicin resistance, and cultured at 28°C, 250 rpm for 18-24 hours; centrifuged at room temperature, 5500 rpm for 10 min; the supernatant was discarded, and the cells were resuspended in an impregnation solution (1 / 2MS 2.2 g / L; sucrose 50 g / L; Silwet L-77 200 μl / L; pH adjusted to 5.7 with NaOH) and adjusted to OD 600 nm is 0.8. The wild-type Arabidopsis Col-0 inflorescence that is about to bloom is completely immersed in the bacterial suspension for 2 minutes; Col-0 is taken out and placed on its side in a moist tray, and after being placed away from light for 24 hours, Col-0 is placed vertically and cultured like ordinary plants, and seeds are harvested to obtain T0 generation IDD7-transfected Arabidopsis seeds. The empty vector plasmid pAMBIA1302-35S-GFP and pCXSN-35S-SRDX are subjected to the same operation as above and used as the control group later.

[0059] 3. Screening and verification of IDD7 transgenic Arabidopsis

[0060] 1) Screening of T1 generation positive transgenic plants

[0061] After disinfection and vernalization at 4°C for three days, the T1 generation seeds were evenly spread on a 1 / 2MS plate containing 25 μg / mL hygromycin and cultured for about a week. The growth of non-positive seedlings was inhibited. Compared with non-positive seedlings, positive seedlings had larger leaves and longer roots. They were transferred to normal culture medium and grown vertically for 3 days, and then transferred to soil. When the seeds matured, individual plants were harvested as T2 generation (i.e., T2 generation IDD7 Arabidopsis seeds).

[0062] 4. Phenotypic analysis of IDD7-transgenic Arabidopsis

[0063] 1) Observation of the growth status of transgenic Arabidopsis seedlings

[0064] The disinfected and vernalized pAMBIA1302-35S-GFP and pAMBIA1302-35S-IDD7-GFP T2 transgenic Arabidopsis seeds were spread on 10 cm MS solid medium (formula: 1 / 2MS salt 2.2 g / L, sucrose 10 g / L, pH 5.8, agar concentration 8 g / L plate), cultured vertically, temperature conditions: 23 ° C, 16 hours of light and 8 hours of darkness. After the plants grew for 8 days, they were observed and photographed. Three-quarters of the pAMBIA1302-35S-IDD7-GFP T2 materials showed yellowing leaves and shortened taproots, and the other 1 / 4 were normal, which was in line with Mendel's segregation ratio. The taproot length was statistically analyzed. The taproot length of the pAMBIA1302-35S-IDD7-GFP T2 transgenic Arabidopsis materials was significantly different from that of the control group (p<0.01). The above experiments were repeated three times independently ( Figure 1 (a, d).

[0065] 2) 35S-IDD7-GFP transgenic material

[0066] The above 1) 35S::IDD7-GFP root tip was placed under a fluorescence microscope to observe the protein expression of IDD7-GFP. The abnormal phenotype of IDD7-GFPT2 material was consistent with the expression of IDD7-GFP. IDD7-GFP was highly expressed in the nuclei of the main root cells of Arabidopsis thaliana ( Figure 1 Middle b) indicates that the phenotype of the functional material obtained above is caused by overexpression of IDD7 family protein fused with GFP.

[0067] When pCXSN-35S-SRDX and pCXSN-35S-IDD7-SRDX T2 were planted on 1 / 2MS medium, 3 / 4 of the materials showed shortened taproots, yellow leaves or no obvious taproot phenotype, and the other 1 / 4 were normal, which was consistent with Mendel's segregation ratio. The taproot length of the pCXSN-35S-IDD7-SRDX T2-transformed Arabidopsis material was significantly different from that of the control group (p<0.01). The above experiments were repeated three times independently ( Figure 1c, e).

[0068] Based on the above results, overexpression of the genes EIDD7-GFP or IDD7-SRDX in plants can inhibit root growth and leaf development. Overexpression of the gene EIDD7 alone inhibits development, and overexpression of the gene EIDD7 after fusion with SRDX can also inhibit development.

[0069] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of AtIDD7 gene in regulating plant growth and development.

2. The use according to claim 1, characterized in that: Overexpression of the AtIDD7 gene negatively regulates plant growth and development.

3. The use according to claim 2, characterized in that: Overexpression of the AtIDD7 gene inhibits plant leaf development.

4. The use according to claim 2, characterized in that: Overexpression of the AtIDD7 gene inhibits plant taproot elongation.

5. The use according to claim 1, characterized in that: The plants include Arabidopsis thaliana.

6. The use according to claim 1, characterized in that: The nucleotide sequence of the AtIDD7 gene is shown in SEQ ID No.

1.

7. The use according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the AtIDD7 gene is shown in SEQ ID No.

2.

8. A method for regulating plant growth and development, characterized in that: The following steps are involved: 1) constructing the AtIDD7 gene in the application of claim 1 into an expression vector containing EGFP or SRDX to obtain a recombinant expression vector; 2) transforming the recombinant expression vector obtained in step 1) into Agrobacterium tumefaciens to obtain transformed bacteria; 3) Infecting plants with the transformed bacteria obtained in step 2) to regulate plant growth and development.

9. The method according to claim 8, characterized in that The expression vector in step 1) includes pAMBIA1302-35S-EGFP or pCXSN-35S-HA-SRDX.

10. The method according to claim 8, characterized in that In step 2), the Agrobacterium tumefaciens is Agrobacterium tumefaciens GV3103; The plants in step 3) include Arabidopsis thaliana, and Arabidopsis thaliana flowers are infected.

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