CN106520722A - Application of casein kinase PPK related to growth and development of plants - Google Patents

Abstract

The invention provides an application of a casein kinase PPK related to the growth and development of plants. The casein kinase PPK is PPK1, PPK2, PPK3 or PPK4, and the sequence registration numbers thereof are At3G13670, At5G18190, At3G03940 and At2G25760 respectively. The casein kinase PPK is used for improving the growth and development of the plants, and then is applied to crop production.

Description

Application of a kind of casein kinase PPK related to plant growth and development

technical field

The invention belongs to the field of biotechnology, and in particular relates to the application of a kind of casein kinase PPK related to plant growth and development.

Background technique

Protein kinases are a class of phosphorylated proteins that use GTP or ATP as phosphate donors to transfer γ-phosphate groups to serine, threonine or tyrosine, thereby regulating related signaling pathways. Eukaryotic protein kinases are a very large superfamily of homologous proteins, including the A-C-G group, the CaMK group, the C-M-G-C group, the conventional protein-casein kinase group, and other protein kinase families (Hanks and Hunter 1995).

In the middle of the 19th century, researchers discovered for the first time a protein kinase capable of phosphorylating casein in rat liver, named casein kinase (Burnett and Kennedy 1954). Since then, casein kinase has attracted widespread attention. At present, compared with the research on casein kinases in animals, the research on casein kinases in plants is relatively preliminary.

Casein kinases are evolutionarily conserved in eukaryotes and regulate a variety of cellular processes by binding or phosphorylating protein substrates, such as membrane trafficking, circadian clock, cancer, DNA-repair pathways (Dhillon and Hoekstra 1994), Wnt signaling, Cytoskeleton maintenance, RNA metabolism, parasite infection, etc. (Knippschild et al. 2005; Liu et al. 2009; Liu et al. 2003; Venerando et al. 2014). However, the function in plants is still less studied. Liu et al. (2003) found that rice casein kinase 1 plays a role in root development and hormone response (Liu et al. 2003).

Contents of the invention

The purpose of the present invention is to provide the application of a class of casein kinase PPK related to plant growth and development, and explore the physiological functions of a class of homologous casein kinases (At3G13670, At5G18190, At3G03940, At2G25760) in Arabidopsis, thereby Improve plant growth and development, and then apply to crop production.

To achieve the above object, the present invention adopts the following technical solutions:

(1) In order to achieve the purpose of the present invention, the present invention first constructs a recombinant plant expression vector, and the recombinant expression vector is ACT2::2*Flag-GFP-linker-PPK .

Among them, the linker is composed of 5 amino acids in series, and its sequence is: PAPAP; GFP is green fluorescent protein.

The construction method of the vector is as follows:

a. The four genes At3G13670, At5G18190, At3G03940 and At2G25760 were found in the Tair database, because this type of protein kinase was obtained when the Arabidopsis blue light receptor CRY2 was screened in the yeast cDNA library in our laboratory, so it was named PPK1, PPK2 , PPK3 and PPK4. PPK is the abbreviation of Photoregulatory Protein Kinases. Design PCR amplification primer pair according to its sequence, as shown in table 1;

b. Using the total cDNA of wild Col-0 Arabidopsis thaliana as a template, the complete sequences of four casein kinases were obtained by PCR;

c. The PCR fragment was connected to the plant expression vector pFGFP, as shown in Figure 1. The sequence is identical to that of the target gene after sequencing;

d. Using the Agrobacterium-mediated method, Arabidopsis rdr6-11 (Peragine et al. 2004) plants were infected by flower dipping. The seeds of transformed plants were collected and screened for herbicide resistance (Basta), and then transgenic positive plants were obtained through western blot verification.

e. Perform phenotypic analysis on the positive transgenic plants, the phenotypes are shown in Figure 4 and Figure 6.

(2) In order to better illustrate the function of PPKs, the present invention also constructed a polygenic interference vector, which is: Act2::amiRPPK1 /Ubq10::amiRPPK4 (Bar) and Act2::amiRPPK2 /Ubq10::amiRPPK3 (Hygromycin).

a. According to the sequences of At3G13670, At5G18190, At3G03940 and At2G25760, artificial microRNAs were designed for them. The nucleotide sequences are shown in Table 2. The backbone of artificial microRNA (amiR) was cloned by referring to the method of Jen sheen's laboratory (Li et al.2013). And amiRPPK1 and amiRPPK4 were linked into the bivalent vector pDT1-Bar, and amiRPPK2 and amiRPPK3 were linked into the bivalent vector pDT1-Hyg. The vector map of pDT1-Bar is shown in Figure 2. The vector map of pDT1-Hyg is shown in Figure 3.

b. After the sequencing was correct, the vectors were respectively transformed into Agrobacterium Agl0. Arabidopsis wild-type Col-0 was co-transformed with Agrobacterium containing Act2 ::amiRPPK1/ Ubq10::amiRPPK4 (Bar) and Act2::amiRPPK2/Ubq10::amiRPPK3 (Hygromycin) interference vectors. The seeds of the transformed plants were collected and screened for herbicide resistance (Basta) and hygromycin double resistance, and then the transgenic positive plants were verified by RT-PCR method, and the transgenic plants that interfered with the four genes at the same time were named as amiR ^4k .

c. Phenotype analysis was performed on the interfering plants, and the phenotypes are shown in Figure 5 and Figure 7.

Table 1. PPK1 , PPK2 , PPK3 and PPK4 primer sequences for cloning

Table 2. Sequences of artificaial microRNAs designed for PPK1 , PPK2 , PPK3 and PPK4

The advantage of the present invention is that: in the present invention, a class of important tyrosine protein kinases is studied, and protein kinases function by regulating different substrates, and these substrates may be in different plant signal transduction pathways and affect the growth and development of plants . Previous studies only started with these single substrates to study their effects on plant growth and development, and the changes in plant traits may not be significant. However, the present invention starts with protein kinase, and changing its expression level can simultaneously change the activity of many of its substrates, thereby affecting different aspects of plant growth and development. The specific phenotypes have been listed in this patent, such as changing the length of plant hypocotyls, leaves, etc. shape and flowering time.

Description of drawings

Fig. 1 is a map of pFGFP vector in the present invention.

Fig. 2 is the vector map of pDT1-Bar in the present invention.

Fig. 3 is the vector map of pDT1-Hyg in the present invention.

Fig. 4 is a comparison of the length of hypocotyls between the transgenic material and the wild type in the present invention. Among them, WT is Arabidopsis rdr6-11 , cry1 is blue light receptor cryptochrome1 deletion mutant, cry2 is blue light receptor cryptochrome2 deletion mutant, cry1cry2 is double mutant, Actin2 ::PPK1 is transgenic line.

Fig. 5 is a comparison of the length of hypocotyls between the interference strain and the wild type in the present invention. Among them, WT is Arabidopsis Col-4, cry1cry2 is a cryptochrome double mutant, phyAphyB is a phytochrome double mutant, and amiR ^4k is a multi-gene interference strain of PPK1, PPK2, PPK3 and PPK4.

Fig. 6 is a comparison of leaf shape between the transgenic material of the present invention and the wild type. Wherein the control is the transgenic plant background strain rdr6-11 , Actin2::PPK1 , Actin2::PPK2, Actin2::PPK3, Actin2::PPK4 are the transgenic lines.

Fig. 7 is a comparison of the flowering time between the interfering strain and the wild type in the present invention. Among them, WT is Arabidopsis Col-4, and amiR ^4k is PPK1, PPK2, PPK3 and PPK4 polygenic interference lines.

detailed description

Example 1

First, PCR amplification primer pairs were designed according to the PPK1 sequence, as shown in Table 1. The complete sequence of PPK1 was obtained by PCR using the total cDNA of wild Arabidopsis thaliana as a template. The PCR fragment was connected to the plant expression vector pFGFP, as shown in Figure 1. After sequencing, the sequence identical to that of the target gene was obtained. Then, Arabidopsis rdr6-11 ( Peragine et al. 2004) plants were infected by flower dipping using the Agrobacterium-mediated method. The seeds of the transformed plants were collected and screened for herbicide resistance (Basta), and then transgene-positive plants were obtained through western blot verification, and finally three expressing transgenic lines were obtained. Using wild-type and PPK transgenic Arabidopsis as materials, they were light-treated (blue light: 30 μmol m-2s-1, red light: 30, far-red light: 2.5, dark), and the hypocotyls were measured after growing for 5 days. The measurement data are shown in Table 3.

Table 3. Effect of PPK1 on Arabidopsis hypocotyl growth (mm)

Note: The same letters on shoulders of peer data indicate no significant difference (p>0.05), and different letters on shoulders indicate significant differences (p<0.05).

After the other three protein kinases were overexpressed, the hypocotyl phenotype was basically the same as that of PPK1, and the hypocotyls treated with light were higher than those of the control group. These four protein kinases have functional redundancy, so not all the hypocotyl phenotype data of the overexpression lines are shown here, but the phenotypes of the polygenic interference lines as described below are shown for further clarification Effects of this class of protein kinases on hypocotyl growth.

The present invention also constructs a multi-gene interference vector, and designs artificial microRNA for it according to the sequences of At3G13670, At5G18190, At3G03940 and At2G25760, and its nucleotide sequence is shown in Table 2. The backbone of artificial microRNA (amiR) was cloned according to the method of Jen sheen's laboratory (Li et al. 2013). And amiRPPK1 and amiRPPK4 were linked into the bivalent vector pDT1-Bar, and amiRPPK2 and amiRPPK3 were linked into the bivalent vector pDT1-Hyg. The vector map of pDT1-Bar is shown in Figure 2. The vector map of pDT1-Hyg is shown in Figure 3.

After the sequencing was correct, the vectors were respectively transformed into Agrobacterium Agl0. Arabidopsis wild-type Col-0 was co-transformed with Agrobacterium containing Act2::amiRPPK1/Ubq10:: amiRPPK4 (Bar) and Act2::amiRPPK2/Ubq10::amiRPPK3 (Hygromycin) interference vectors. The seeds of the transformed plants were collected and screened for herbicide resistance (Basta) and hygromycin double resistance, and then the transgenic positive plants were verified by RT-PCR method, and the transgenic plants that interfered with the four genes at the same time were named as amiR ^4k .

The wild-type and ppk-disturbed strains were used as materials, and subjected to light treatment (blue light: 20, red light: 20, far-red light: 2.5, dark), and the hypocotyls were measured after growing for 5 days. The measurement data are shown in Table 4. Contrary to the PPK overexpression phenotype, the hypocotyls of the ppk interference lines were shorter than those of the wild type under light treatment.

Table 4. Hypocotyl growth changes of PPK interference lines (mm)

Note: The same letters on shoulders of peer data indicate no significant difference (p>0.05), and different letters on shoulders indicate significant differences (p<0.05).

The wild-type and ppk-disturbed strains were used as materials, and the flowering time was counted under long-day conditions (16h light/8h dark). The statistical data are shown in Table 5. The ppk disturbed lines showed delayed flowering time under long-day conditions.

Table 5. Changes in flowering time of PPK interference strains

Note: The same letters on shoulders of peer data indicate no significant difference (p>0.05), and different letters on shoulders indicate significant differences (p<0.05).

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

SEQUENCE LISTING

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Claims (2)

1. the class casein kinase PPK related to growth and development of plants is changing plant hypocotyl length, blade shape and opens Application in taking time.

2. application according to claim 1, it is characterised in that：Described casein kinase PPK is PPK1, PPK2, PPK3 Or PPK4, its Sequence accession number is respectively At3G13670, At5G18190, At3G03940 and At2G25760.