CN112830962A - Apple pillar type candidate gene Co41 related protein and coding gene and application thereof - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, and discloses an apple pillar type candidate gene Co41 related protein, and a coding gene and application thereof. The sequence of the apple column type candidate gene Co41 related protein is shown in SEQ ID NO. 2. The invention identifies a new apple columnar related gene and provides evidence for revealing the phenotypic mechanism of columnar apples. The gene over-expression mode plant obviously promotes the enlargement of leaves and the increase of chlorophyll content, and is beneficial to enhancing the photosynthetic capacity of the plant. The invention can be applied to the genetic improvement of apples or other woody plants, has important theoretical value and production and application potential for cultivating high-light-efficiency leaf curtains of fruit trees and improving photosynthetic capacity.

Description

Apple pillar type candidate gene Co41 related protein and coding gene and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an apple column type candidate gene Co41 related protein and a coding gene and application thereof.

Background

Apple (Malus domestica Borkh.) is a plant of Malus genus of Maloideae subfamily of Rosaceae, and its tree is deciduous tree. The apple fruits are rich in minerals and vitamins and are one of the three fruits in China. With the improvement of living standard of people, the demand and the quality of fruits are more emphasized, so that the improvement of the single yield and the quality are the main problems to be solved at present under the trend that the planting area of apple trees is gradually saturated. Researches show that the difference of the photosynthesis intensity of the apple trees has great influence on yield and quality, and has very important significance for cultivating ideal apple trees.

Apple tree leaf size and thickness also have a large impact on apple yield. Wherein the leaves of the apple pillar type variety are larger than those of the non-pillar type. The apple columnar variety ` Weisaike ` (McIntosh Wijcik) was derived from the bud mutation of non-columnar apples ` Asu ` (McIntosh) (Lapins,1974), discovered in 1961 by Anthony Wijcik in an apple orchard in British Columbia, Canada, and named after the name of the discoverer. From this point on, 'Wencek' and its filial generation become columnar apples. The growth characteristics of the columnar apples are represented as thick and strong branches, short internodes, high germination rate, more short branches on the main trunk, less long branches and small branch angle. Genetic analysis of the hybrid population showed compliance with the mendelian quality trait inheritance rule, controlled by the dominant gene Co (Dai et al, 2003). Selecting Malus x domestica v1.0 in GDR to search the predicted gene and the selectable gene in the Co region, selecting the gene in 18.51-19.1 Mb region of apple chromosome 10, eliminating the gene outside the region, and recording the function, size and corresponding position of the predicted gene. The genes outside the deleted region were deleted to obtain 64 predicted genes (Nos. 1 to 64). The 64 predicted genes obtained preliminarily were analyzed at the transcriptional level. Searching through SRA (stem tip, leaf, root and fruit) and EST (taxi: 3750) databases of the apple, comparing and analyzing the searched result sequence and the genome, searching 32 genes in total, predicting that the genes have various functions and are expressed in different tissues and organs of the apple. Wherein, the No. 56 and No. 60 genes are only expressed in roots; genes No. 1 and No. 20 are expressed in fruit, leaves and roots; genes No. 4, No. 43 and No. 62 are expressed in fruits, roots and stem tips; genes 33, 41 and 51 are expressed in fruits, leaves and stem tips; gene No. 26 is expressed in roots and shoot tips; gene No. 31 is expressed in leaves and shoot tips; gene number 40 is expressed in leaves and roots; no. 6, No. 9, No. 10, No. 11, No. 12, No. 13, No. 14, No. 27, No. 28, No. 30, No. 32, No. 36, No. 37, No. 38, No. 44, No. 48, No. 52, No. 54, No. 64 genes are expressed in all of fruits, leaves, roots and stem tips. The 32 prediction genes retrieved were used as primary screening genes for subsequent studies.

Among 29 predicted genes amplified to a cDNA sequence, qRT-PCR specific primers are designed by utilizing the cloned gene sequence, and the expression difference of the genes in stem tip tissues of columnar apple 'Weisai' and non-columnar apple 'Xu' is analyzed. The results show that 29 rescreened genes are expressed in stem tip tissues of both columnar apple 'wesseck' and non-columnar apple 'asahi'. In the shoot tip tissue of the columnar apple 'wesseck', 10 predicted genes are up-regulated, and 19 genes are down-regulated. Of the genes whose expression was upregulated, 6 genes were upregulated significantly, including genes No. 26 (RNA-directed DNA polymerase activity), No. 41 (PPR family), No. 33 (involved in carbohydrate synthesis), No. 38 (containing the 2OG-Fe (ii) oxygenase domain), No. 28 (autophagy protein 9 family) and No. 32 (function unknown). The up-regulation times are 62 times, 34.8 times, 4.7 times, 14 times, 2.1 times and 2 times in turn. Among the genes whose expression was down-regulated, 9 genes were significantly down-regulated, including genes No. 30 (function unknown), No. 9 (phosphate transmembrane transporter activity), No. 37 (transferase activity), No. 6 (function unknown), No. 44 (containing 2OG-Fe (ii) oxygenase domain), No. 10 (actin binding function), No. 27 (function unknown), No. 11 (DUF647 protein family), and No. 4 (MYB transcription factor family). The down-regulation times are 18 times, 4.1 times, 2.9 times, 2.8 times, 2.3 times, 2.1 times, 1.8 times, 1.6 times and 1.6 times in sequence.

The genome of apple has been published in 2010, but the functions of a plurality of genes are not clear, and the research on the gene functions by using an overexpression technology becomes an effective and rapid method. The transgenic plant obtains a certain phenotype by over-expressing the gene into tobacco, has certain feasibility, short time consumption and convenient operation. Therefore, the method for searching new functional genes and clarifying the functions of the genes has important theoretical and practical significance.

On apples, direct functional verification technology of genes is mature, the genes of the apples are generally over-expressed by adopting an agrobacterium-mediated 'Gala' leaf disc method, the period is longer than that of model plants due to the characteristics of woody plants, the transformation efficiency is lower than that of the model plants, but later-stage phenotypes of the apples can be expected according to the current verification means. Apple gene heterologous expression pattern plants have been reported to characterize their function, for example, transcription factor MdDREB76 was overexpressed in tobacco to verify their response to drought and salt damage (Sharma et al.2019); MdMYB1 and MdbHLH3 were over-expressed in tobacco and verified to be associated with anthocyanin synthesis (Xie et al.2017); MdSUP11 overexpressing tobacco was shown to be associated with floral development (Xu et al.2018).

Disclosure of Invention

In view of the above, the present invention aims to provide an apple pillar type candidate gene Co 41-related protein and a coding gene thereof, such that the related protein can significantly promote plant leaf enlargement and thickening, increase chlorophyll content, thereby enhancing plant photosynthetic capacity and increasing yield;

it is another object of the present invention to provide an expression cassette, an expression vector and an expression host capable of expressing the protein of interest;

the invention also aims to provide the application of the related protein and the coding gene thereof or the expression cassettes, the expression vectors and the expression hosts thereof in optimizing the size and the thickness (increasing and thickening) of plant leaves and the chlorophyll content (increasing);

the invention also aims to provide the related protein, the coding gene thereof or the expression cassettes, the expression vectors and the expression hosts thereof, and application of the related protein, the coding gene, the expression cassettes, the expression vectors and the expression hosts in cultivation of transgenic plants with large and thick leaf traits and high chlorophyll content traits.

In order to achieve the above purpose, the invention provides the following technical scheme:

the sequence of the related protein is shown as SEQ ID NO. 2, the protein is obtained from columnar apple 'Mcintosh Wijcik', the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1 in the invention, the coding gene of the apple columnar candidate gene Co41 related protein also comprises other genes taking the sequence shown as SEQ ID NO. 1 as an exon, and for example, the sequence shown as SEQ ID NO. 1 is divided into a plurality of exons and other intron constitutive genes.

Herein, the apple pillar candidate gene Co 41-related protein may be non-natural, e.g., synthetic or expressed from an artificial vector. The term "non-natural" means that the target substance is not naturally occurring in nature, which does not preclude the non-natural substance from having the same structure and/or composition as the naturally occurring substance.

Experiments prove that a column-type related Co41 gene is screened from a column-type apple 'Mcintosh Wijcik', the Co41 gene is introduced into Nicotiana benthamiana through an agrobacterium transformation method, leaves of an obtained transgenic strain are obviously increased and thickened, and the chlorophyll content is obviously increased, so that the function of a function-unknown protein, namely a column-type candidate gene Co41 related protein of the apple, is verified.

Based on the superiority of the technical effects, the invention provides the application of the apple columnar candidate gene Co41 related protein and the coding gene thereof in optimizing the size and thickness of plant leaves and the chlorophyll content, or in cultivating transgenic plants with the characteristics of large and thick leaves and high chlorophyll content. In a specific embodiment of the present invention, the plant of the invention is preferably apple or tobacco, since the function of the protein is verified by the model plant Nicotiana benthamiana according to the consistent verification means in the art, and the later phenotype of apple can be expected according to the current verification means.

In addition, the invention also provides an expression cassette, a recombinant expression vector or a recombinant expression host containing the coding gene of the apple columnar candidate gene Co 41-related protein, and application of the expression cassette, the recombinant expression vector or the recombinant expression host in optimizing the size and the thickness of plant leaves and the chlorophyll content, or application in cultivating transgenic plants with the characteristics of large and thick leaves and high chlorophyll content. The plant is preferably apple or tobacco.

Preferably, the expression cassette is an expression element with a promoter, a target gene and a terminator, and other gene elements for assisting expression can be added according to requirements; the recombinant expression vector is preferably an over-recombinant expression vector, in the specific embodiment of the invention, the over-expression vector is a commercially available pCAMBIA1305, and the coding gene of the apple columnar candidate gene Co41 related protein can be inserted between enzyme cutting sites of the vector pCAMBIA1305 to construct the over-expression vector, for example, between enzyme cutting sites NcoI and BsteII; the recombinant expression host is preferably a recombinant engineering bacterium, including but not limited to agrobacterium, saccharomyces cerevisiae, escherichia coli and the like. When the coding gene of the apple pillar candidate gene Co 41-related protein is used for constructing a recombinant vector, a recombinant expression host, a transgenic plant or an expression cassette, enzyme cutting sites can be added at the tail end of the apple pillar candidate gene according to the conventional technology in the field.

According to the application, the invention provides a method for cultivating transgenic plants with large and thick leaf traits and high chlorophyll content traits, and the transgenic plants are obtained by over-expressing coding genes of proteins related to apple columnar candidate gene Co41 in starting plants.

More specifically, an overexpression vector containing an encoding gene of a protein related to apple columnar candidate gene Co41 is constructed and is introduced into a starting plant to obtain the transgenic plant. Overexpression vectors can be integrated using the T4 ligation method.

According to the technical scheme, the invention identifies a novel apple columnar related gene and provides evidence for revealing a columnar apple phenotype mechanism. The gene over-expression mode plant obviously promotes the enlargement of leaves and the increase of chlorophyll content, and is beneficial to enhancing the photosynthetic capacity of the plant. The invention can be applied to the genetic improvement of apples or other woody plants, has important theoretical value and production and application potential for cultivating high-light-efficiency leaf curtains of fruit trees and improving photosynthetic capacity.

Drawings

FIG. 1 shows a map of the vector pCAMBIA1305-Co41 according to the present invention;

FIG. 2 is a photograph showing transgenic tobacco of the T2 generation growing for 90 days; wherein c-e transgenic tobacco, a and b are wild type contrast; 1cm on a scale;

FIG. 3 is a statistical plot of leaf area for 90 days of T1 generation wild-type tobacco and transgenic tobacco growth; MdCo 41: overexpression lines, WT: wild type, pCAMBIA 1305: empty plasmid tobacco is transferred;

FIG. 4 is a statistical plot showing chlorophyll a, chlorophyll b and carotene contents of wild type tobacco and transgenic tobacco of the T1 generation; line1-3 MdCo41 overexpression line, WT: wild type, pCAMBIA 1305: empty plasmid tobacco was transferred.

Detailed Description

The invention discloses an apple columnar candidate gene Co41 related protein and a coding gene and application thereof, and can be realized by appropriately improving process parameters by taking the contents of the protein as reference by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the method and its application have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the invention may be practiced and applied by modifying or appropriately combining the methods and applications described herein without departing from the spirit and scope of the invention.

If not specifically stated, the reagents, carriers and other materials used in the invention can be synthesized and purchased by reagent companies, the columnar apple 'McIntosh Wijcik' is planted at the Shanzhuang laboratory station of China university of agriculture, and the Nicotiana benthamiana is planted in the glass greenhouse of China university of agriculture.

Three repeated experiments are set in the quantitative experiment, and the results are averaged.

The invention is further illustrated by the following examples.

Example 1: cloning of Co41 Gene

1. Specific primer pairs (F and R) were designed, synthesized by Biotech:

Co41F:5’-ATGTCAACTGTTGTTTCGGCTTGTG-3’；

Co41R:5’-TTAAAAACTGCACCACCATAACTCAGAGTC-3’。

2. the CTAB method extracts RNA of apple 'Wijcik' stem tip and carries out reverse transcription to obtain cDNA.

3. And (3) taking the cDNA in the step (2) as a template, carrying out PCR amplification on the F and the R by using the specific primers in the step (1), and recovering PCR amplification products.

4. Sequencing the PCR amplification product of step 3.

The sequencing result is sequence 1 in the sequence table, the gene is named as Co41 gene, the amino acid sequence of the protein is shown as sequence 2 in the sequence table, and the protein consists of 224 amino acid residues. The invention designs NcoI restriction enzyme cutting site at the 1 st-6 th nucleotide of the 5' end of the sequence 1, designs the nucleotide sequence of BsteII restriction enzyme cutting site at the 673 rd-679 th nucleotide, and is convenient for the subsequent vector construction.

5. The PCR product is inserted into a pMD18T-simple vector (purchased from TaKaRa bioengineering company) to obtain a vector pMD18T-simple-Co41, and sequencing is carried out, so that the vector pMD18T-simple-Co41 is a cloning vector obtained by inserting a sequence 1 in a sequence table into a pMD18T-simple vector.

Example 2: acquisition and functional study of transgenic tobacco

1. Construction of recombinant vector (pCAMBIA1305-Co41)

(1) The vector pMD18T-simple-Co41 was double-digested with restriction enzymes NcoI and BsteII, and the small fragment was recovered.

(2) The vector pCAMBIA1305 (stored in the laboratory) was digested with restriction enzymes NcoI and BsteII, and the vector backbone was recovered.

(3) And (3) connecting the small fragment in the step (1) with the vector skeleton in the step (2) by using T4 ligase to obtain a ligation product, transforming the ligation product into escherichia coli DH5 alpha to obtain a transformant, extracting a plasmid of the transformant, sending the plasmid to sequencing, and obtaining a recombinant vector (a plasmid map is shown in figure 1) by inserting the sequence 2 in the sequence table between NcoI and BsteII enzyme cutting sites of pCAMBIA1305, wherein the plasmid is named pCAMBIA1305-Co 41.

2. Acquisition of transgenic Positive seedlings

(1) pCAMBIA1305-Co41 transformation of Agrobacterium EHA105

Adding 1 μ g of the obtained pCAMBIA1305-Co41 into 200 μ l of EHA105 competent cell, mixing, and standing for 5 min; quickly freezing in liquid nitrogen for 1min, water bathing at 37 deg.C for 5min, adding 1ml YEB liquid culture medium, and performing shake culture at 28 deg.C and 150rpm for 4 hr; centrifuging at 5000rpm for 3min, discarding supernatant, adding 0.1ml YEB liquid culture medium, and suspending cells again; the resulting suspension was plated on YEB solid plates containing 50. mu.g/ml kanamycin and 50. mu.g/ml rifampicin, and cultured at 28 ℃ for about 48 hours to obtain transformants. Carrying out PCR identification on the transformant by using bacterial liquid and primers used for identification as follows:

Co41F:5’-ATGTCAACTGTTGTTTCGGCTTGTG-3’；

Co41R:5’-TTAAAAACTGCACCACCATAACTCAGAGTC-3’。

the results showed that a fragment of approximately 672bp was obtained, demonstrating that pCAMBIA1305-Co41 has been successfully transferred into EHA 105. The transformant containing pCAMBIA1305-Co41 was named recombinant Agrobacterium EHA105/pCAMBIA1305-Co 41.

(2) Transformed tobacco

Inoculating recombinant Agrobacterium EHA105/pCAMBIA1305-Co41 in 10ml YEB liquid culture medium containing 50 ug/ml kanamycin and 50 ug/ml rifampicin, culturing overnight at 28 deg.C under shaking at 200 rpm;

secondly, inoculating the strain in 100ml YEB culture medium containing the same antibiotic at a volume ratio of 1:100 (volume ratio) one day before transformation, carrying out amplification culture until OD600 is 0.7-0.8, centrifuging at 7000rpm for 5min, collecting the strain, and suspending the strain in an infiltration buffer solution (comprising sucrose, MS powder and acetosyringone, wherein the mass percentage of the sucrose is 3 percent of the sucrose, the MS powder is 4.404g/l and the acetosyringone is 100 mu mol/l), so that OD600 is 0.5-0.6, namely the recombinant Agrobacterium tumefaciens EHA105/pCAMBIA1305-Co41 bacterial solution;

thirdly, transforming agrobacterium into tobacco leaves by adopting a leaf disc method:

1) the genetic transformation of tobacco needs to be carried out in ultra-clean, tender tobacco leaves are clamped by tweezers, the leaf edges and the main veins of the tobacco leaves are cut off, and the leaves are cut into small blocks with the side length of about 1cm for standby infection.

2) Laying a layer of sterile filter paper on the co-culture medium in advance, clamping the cut tobacco leaf discs by using sterile tweezers, putting the tobacco leaf discs into a plate or a conical flask containing a proper amount of agrobacterium infection liquid, carrying out oscillation infection for about 10 minutes, sucking residual liquid by using the sterile filter paper, and transferring the tobacco leaf discs to the co-culture medium containing AS for 3 days in the dark.

3) After 3 days of dark culture, the tobacco leaves are transferred to a culture medium containing Hyg, dark screening culture is carried out for about three weeks, and then the tobacco leaves are transferred to light screening culture.

4) And (3) replacing the screening culture medium every 15 days or so until the callus at the wound of the explant grows resistant buds (2-3 cm), cutting and inserting the callus into a rooting culture medium, and performing rooting culture.

5) The media used for tobacco transformation were as follows:

co-culture medium: MS minimal medium +2.5 mg/L6-BA +0.2mg/L IAA +50 mu M AS

Screening a culture medium: MS +2 mg/L6-BA +0.2mg/L IAA +20mg/LHyg +250mg/L Cef

Rooting culture medium: MS minimal medium +0.1mg/L IAA +20mg/LHyg +250mg/L Cef

(3) Molecular characterization of transgenic positive resistant shoots

(ii) detection of DNA level

Collecting leaves of the rooted resistant plants, extracting DNA and RNA of the leaves, taking a plasmid pCAMBIA1305-Co41 as a positive control, taking DNA of unloaded and wild tobacco as a negative control, and carrying out PCR amplification by using the following primers:

Co41F:5’-ATGTCAACTGTTGTTTCGGCTTGTG-3’；

Co41R:5’-TTAAAAACTGCACCACCATAACTCAGAGTC-3’。

PCR System (25.0 ul): 10 XEx Taq PCR Buffer 2.5ul, dNTP (25mM)2.0ul, 5 'primer (5pmol/ul)1.0ul, 3' primer (5pmol/ul)1.0ul, ExTaq enzyme (5U/ul)12.5ul, template (1ug/ul)1.0ul, ddH₂O 5ul。

The PCR procedure was: a first round: denaturation at 94 deg.C for 5 min; and a second round: denaturation at 94 ℃ for 50sec, renaturation at 52 ℃ for 50sec, extension at 72 ℃ for 70s, 30 cycles; and a third round: extension at 72 ℃ for 10 min.

(4) Functional study

Firstly, after the T2 generation transgenic tobacco grows for 90 days, the phenotype is compared with that of starting tobacco growing for the same days, and the increase and thickening of tobacco leaves can be visually seen through the graph shown in FIG. 2;

(II) after the T1 generation wild tobacco, the transgenic plasmid tobacco and the transgenic tobacco grow for 90 days, the leaf area is counted, statistical analysis is carried out by SPSS software, and the result is shown in figure 3, wherein the leaf area of the transgenic tobacco obtained by the invention is remarkably increased (P is less than 0.01);

and thirdly, after the T1 generation wild tobacco, the transgenic plasmid tobacco and the transgenic tobacco grow for 90 days, the chlorophyll and carotene contents are counted, and the result is shown in figure 4, and the chlorophyll a, chlorophyll b and carotene contents of the transgenic tobacco obtained by the invention are remarkably improved (P is less than or equal to 0.001).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of agriculture in China

<120> apple pillar type candidate gene Co41 related protein and encoding gene and application thereof

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atggaagtgc tcggagttca cggatatgca aatgaagcac cagcaatggt ctgcaagatg 60

gagagagaga agatcaagcc aaatggggtt acttttgaaa gtgttttaag tgcctgcact 120

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Glu Ser Val Leu Ser Ala Cys Thr His Ser Gly Phe Val Glu Glu Gly

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Claims (10)

1. The apple column type candidate gene Co41 related protein is characterized in that the protein sequence is shown in SEQ ID NO. 2.

2. The apple pillar-shaped candidate gene Co 41-related protein coding gene of claim 1.

3. The encoding gene of claim 2, wherein the nucleotide sequence is represented by SEQ ID NO. 1.

4. The application of the apple pillar type candidate gene Co41 related protein and the coding gene thereof in optimizing the size and thickness of plant leaves and the chlorophyll content or in cultivating transgenic plants with the characteristics of large and thick leaves and high chlorophyll content.

5. The use of claim 4, wherein the plant is apple or tobacco.

6. An expression cassette, recombinant expression vector or recombinant expression host comprising the coding gene of claim 2 or 3.

7. The use of the expression cassette, recombinant expression vector and recombinant expression host of claim 6 for optimizing plant leaf size and thickness and chlorophyll content, or for breeding transgenic plants with large, thick leaf traits and high chlorophyll content traits.

8. The use of claim 7, wherein the plant is apple or tobacco.

9. A method for breeding a transgenic plant having a large and thick leaf trait and a high chlorophyll content trait, wherein the transgenic plant is obtained by overexpressing the coding gene of claim 2 or 3 in a starting plant.

10. The method according to claim 9, wherein the transgenic plant is obtained by constructing an overexpression vector containing the coding gene of claim 2 or 3 and introducing the overexpression vector into a starting plant.

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