Application and method of atidd7 gene in regulating plant growth and development

Abstract

The present application relates to the field of biotechnology, in particular to the application and method of AtIDD7 gene in regulating plant growth and development, and discloses the application of AtIDD7 protein in regulating plant growth and development for the first time through the research on AtIDD7 protein in plants. The present application can regulate the growth and development of the aboveground and underground parts of plants by overexpressing AtIDD7 protein in plants, and provides excellent gene resources for the improvement of plant growth and development.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application and methods of the AtIDD7 gene in regulating plant growth and development. Background Technology

[0002] The IDD (INDETERMINATEDOMAIN) family of genes encodes proteins that are conserved transcription factors in plants. They were first defined by the ID domain of the maize flowering conversion gene INDETERMINATE1 (ID1), and encode proteins with two canonical (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 various plants, including Arabidopsis thaliana, rice, and maize (Kumar et al. 2019; Colasanti et al. 2006).

[0003] The Arabidopsis IDD family genes are involved in multiple processes, including plant development, sugar homeostasis, gravity response, and regulation of plant hormones (Kumar et al. 2019; Coelho et al. 2018). IDD1 / ENY regulates seed development by influencing ABA homeostasis through GA (Feurtado et al., 2011); IDD2 / GAF1 regulates seed germination in GA-dependent ways (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 morphogenesis and gravity response of aboveground organs by promoting auxin synthesis and transport (Cui et al. 2013). Another study showed that AtIDD15 regulates the gravity response of Arabidopsis inflorescence stems under high temperatures by producing different spliceosomes (Kim et al. 2016). The functions of the genes IDD7, IDD11, IDD12, and IDD13 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] To address the aforementioned issues, this invention provides the application and method of the AtIDD7 gene in regulating plant growth and development. By overexpressing the AtIDD7 gene in plants, this invention can regulate the growth and development of both above-ground and below-ground parts of plants, providing an excellent gene resource for improving plant growth and development.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of the 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 taproot elongation in plants.

[0011] Preferably, the plant includes Arabidopsis thaliana.

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

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

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

[0015] 1) Construct 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) Transform the recombinant expression vector obtained in step 1) into Agrobacterium tumefaciens to obtain the transformed bacteria;

[0017] 3) The transforming bacteria obtained in step 2) are used to infect plants 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] Step 3) The plant includes Arabidopsis thaliana, and Arabidopsis thaliana inflorescence is soaked.

[0021] The beneficial effects of this invention are:

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

[0023] This invention, by overexpressing the AtIDD7 protein in plants, can regulate the growth and development of both above-ground and underground parts of plants, providing an excellent genetic resource for improving plant growth and development. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0025] Figure 1 a) Phenotypes of 35S:EGFP and 35S:EIDD7-GFP T2 generation materials grown on 1 / 2 MS medium on day 8; b) Protein localization of EIDD7-GFP in the main root of Arabidopsis thaliana; c) Phenotypes of 35S:SRDX and 35S:IDD7-SRDXT2 generation materials grown on 1 / 2 MS medium on day 8; d) Main root length statistics of 35S:EGFP and 35S:EIDD7-GFP T2 generation materials grown on 1 / 2 MS medium on day 8; e) Main root length statistics of 35S:SRDX and 35S:IDD7-SRDXT2 generation materials grown on 1 / 2 MS medium on day 8. Detailed Implementation

[0026]

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

[0028] MMMNRDILFHQQQQQQMEENMSNLTSASGDQASVSSGNRTETSGSNINQHHQEQCFVPQSSLKRKRNQPGNPDPEAEVMALSPKTLMATNRFICEVCNKGFQRDQNLQLHKRGH NLPWKLKQRSNKDVVRKKVYVCPEPGCVHHHPSRALGDLTGIKKHFFRKHGEKKWKCEKCSKKYAVQSDWKAHAKTCGTKEYKCDCGTLFSRRDSFITHRAFCDALAEESARAM PNPIMIQASNSPHHHHHQTQQNIGFSSSSQNIISNSNLHGPMKQEESQHHYQNIPPWLISSNPNPNGNNGNLFPPVASSVNTGRSSFPHPSPAMSATALLQKAAQMGSTKSTTP EEEERSSRSSYNNLITTTMAAMMTSPPEPGFQDYYMMNHQHHGGGEAFNGGFVPGEEKNDVVDDGGGETRDFLGLRSLMSHNEILSFANNLGNCLNTSATEQQQQQHSHQD.

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

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

[0031] 1) Construct 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) Transform the recombinant expression vector obtained in step 1) into Agrobacterium tumefaciens to obtain the transformed bacteria;

[0033] 3) The transforming bacteria obtained in step 2) are used to infect plants to regulate plant growth and development.

[0034] This invention constructs the AtIDD7 gene from the application described in the above technical solution into an expression vector containing EGFP or SRDX to obtain a recombinant expression vector. In this invention, the expression vector preferably includes pAMBIA1302-35S-EGFP or pCXSN-35S-HA-SRDX. This invention does not specifically limit the method for constructing the AtIDD7 gene into an expression vector containing EGFP or SRDX; conventional gene construction methods into vectors are acceptable.

[0035] In this invention, the obtained recombinant expression vector is transformed into *Agrobacterium tumefaciens* to obtain the transformed bacteria. In this invention, *Agrobacterium tumefaciens* GV3103 is preferably used. This invention does not have any particular limitation on the method for transforming the recombinant expression vector into *Agrobacterium tumefaciens*; conventional methods are acceptable.

[0036] This invention uses transforming bacteria to infect plants, thereby regulating plant growth and development. In this invention, the plant preferably includes Arabidopsis thaliana, and the inflorescence of Arabidopsis thaliana is infecting the plants. This invention does not specifically limit the method of infecting plants with transforming bacteria; conventional methods can be used by those skilled in the art.

[0037] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1

[0039] Application and regulation methods of the 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 the 35S:EIDD7-GFP and 35S:IDD7-SRDX recombinant vectors

[0042] To obtain plants overexpressing the IDD7 gene, the expression vectors 35S:EIDD7-GFP and 35S:IDD7-SRDX, which are driven by the CaMV 35S (Cauliflower mosaicvirus 35S) promoter, were used for plant transformation.

[0043] The CDS (with stop codon removed) sequence of IDD7 was obtained from the Tair website (https: / / www.arabidopsis.org / ) using the gene ID. The BglII restriction sites of pCAMBIA1302-35S-EGFP and pCXSN-35S-HA-SRDX were selected, and 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, yielding a 1365bp PCR product. The pAMBIA1302-35S-GFP and pCXSN-35S-HA-SRDX plasmids were digested using the BglII single enzyme digestion method. The CDS sequences were then seamlessly ligated into the target vectors using the In-Fusion method. Sequencing revealed the IDD7 gene sequence shown in Sequence 2, resulting in the pAMBIA1302-35S-IDD7-GFP and pCXSN-35S-IDD7-SRDX plasmids, confirming the correct vector construction.

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

[0054] 2. Obtaining the IDD7 gene transgenic Arabidopsis thaliana

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

[0056] The method involved adding the recombinant plasmid to 100 μl of Agrobacterium competent cells, incubating on ice for 5 min, freezing in liquid nitrogen for 20 s, and then incubating at 37°C for 5 min. 700 μl of antibiotic-free LB medium was added, and the cells were incubated at 28°C and 200 rpm for 3 h. The cells were then centrifuged at 3000 rpm, the supernatant was discarded, and 100 μl of LB medium was used to resuspend the cells. This resuspended cells were then plated on LB resistant plates (containing 50 μg / ml kanamycin) and incubated upside down at 28°C. Simultaneously, the empty vector plasmids pAMBIA1302-35S-GFP and pCXSN-35S-SRDX (without the IDD7 gene incorporated) were subjected to the same procedure and subsequently used as control groups.

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

[0058] Single colonies were selected and cultured in 2 ml of LB liquid medium containing kanamycin and rifampicin resistance at 28°C and 250 rpm for 16 hours. 0.2 ml of the bacterial culture was added to 100 ml of LB liquid medium containing kanamycin and rifampicin resistance and cultured at 28°C and 250 rpm for 18-24 hours. The culture was then centrifuged at 5500 rpm for 10 min at room temperature. The supernatant was discarded, and the bacterial resuspended in the infiltration solution (1 / 2 MS 2.2 g / L; sucrose 50 g / L; Silwet L-77 200 μl / L; pH adjusted to 5.7 with NaOH) and adjusted to OD0.05. 600 The nm value was 0.8. Wild-type Arabidopsis thaliana Col-0 inflorescences, nearing flowering, were completely immersed in the bacterial suspension for 2 minutes. Col-0 was then removed, placed sideways on a moistened tray, and kept in the dark for 24 hours. Afterward, Col-0 was upright and cultured like ordinary plants, and seeds were harvested to obtain T0 generation IDD7 Arabidopsis thaliana seeds. The empty vector plasmids pAMBIA1302-35S-GFP and pCXSN-35S-SRDX underwent the same procedure and were subsequently used as a control group.

[0059] 3. Screening and validation of IDD7 transgenic Arabidopsis thaliana

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

[0061] After sterilization and vernalization at 4℃ for three days, T1 generation seeds were evenly spread on 1 / 2 MS plates containing 25 μg / mL hygromycin and cultured for about one week. The growth of non-positive seedlings was inhibited, while positive seedlings had larger leaves and longer roots compared to non-positive seedlings. These were then transferred to normal culture medium and allowed to grow vertically for three days before being transferred to soil. Once the seeds matured, individual plants were harvested as T2 generation (i.e., T2 generation transformed into IDD7 Arabidopsis thaliana seeds).

[0062] 4. Phenotypic analysis of Arabidopsis thaliana with IDD7

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

[0064] Seeds of Arabidopsis thaliana transgenic from pAMBIA1302-35S-GFP and pAMBIA1302-35S-IDD7-GFP T2 generations, after sterilization and vernalization, were sown on 10cm MS solid medium plates (formulation: 1 / 2 MS salt 2.2g / L, sucrose 10g / L, pH 5.8, agar concentration 8g / L). The plates were vertically incubated at 23℃ with a 16-hour light-8-hour dark cycle. After 8 days of growth, the plants were observed and photographed. Three-quarters of the pAMBIA1302-35S-IDD7-GFP T2 transgenic materials showed yellowing leaves and shortened taproots, while the remaining quarter were normal, consistent with Mendelian segregation ratios. Taproot length was statistically analyzed, and the taproot length of the pAMBIA1302-35S-IDD7-GFP T2 transgenic Arabidopsis materials was significantly different from the control group (p<0.01). The above experiments were independently repeated three times. Figure 1 (a, d)

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

[0066] The protein expression of IDD7-GFP was observed under a fluorescence microscope after placing the 1)35S::IDD7-GFP root tip material under a fluorescence microscope. The abnormal phenotype of IDD7-GFPT2 material was consistent with the expression of IDD7-GFP, and IDD7-GFP was highly expressed in the nuclei of Arabidopsis thaliana primary root cells. Figure 1 (b) indicates that the phenotype of the functional material obtained above is due to the overexpression of GFP fused with the IDD7 family protein.

[0067] When pCXSN-35S-SRDX and pCXSN-35S-IDD7-SRDX T2 were seeded on 1 / 2 MS medium, three-quarters of the materials showed shortened taproots, yellowing leaves, or no obvious taproot phenotype, while the other quarter were normal, consistent with Mendelian segregation ratios. Taproot length was statistically analyzed, and the taproot length of the pCXSN-35S-IDD7-SRDX T2 generation Arabidopsis thaliana was significantly different from the control group (p<0.01). The above experiments were independently repeated three times. Figure 1c, e).

[0068] In summary, overexpression of the genes EIDD7-GFP or IDD7-SRDX in plants can inhibit root growth and leaf development. Overexpression of EIDD7 alone inhibits development, and overexpression of EIDD7 fused with SRDX can also inhibit development.

[0069] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. overexpression AtIDD7 application of a gene in inhibiting leaf development of Arabidopsis thaliana or elongation of the main root of Arabidopsis thaliana, characterized in that, The AtIDD7 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. A method of inhibiting leaf development in Arabidopsis thaliana or elongation of the primary root of Arabidopsis thaliana, characterized in that, comprising the following steps: 1) the use according to claim 1 wherein AtIDD7 the gene is constructed into an expression vector containing EGFP to obtain a recombinant expression vector; 2) transforming the recombinant expression vector obtained in step 1) into Agrobacterium tumefaciens to obtain transformed bacteria; 3) infiltrating Arabidopsis thaliana with the transformed bacteria obtained in step 2); The Agrobacterium tumefaciens in step 2) is Agrobacterium tumefaciens GV3103; The infiltrated in step 3) is Arabidopsis thaliana inflorescences.

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