CN113943744B - Application of RCA gene of cymbidium floribundum and vector construction method thereof - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and specifically discloses the application of the RCA gene of the variegated orchid and a vector construction method thereof. The RCA gene can be expressed normally by transferring the RCA of the variegated orchid into Dendrobium officinale, and the overexpression of the RCA gene can increase the transgene The polysaccharide content in Dendrobium officinale, the nucleotide sequence of the RCA gene is shown in SEQ ID NO: 1, and the overexpression of the RCA gene can greatly improve the protein content, the soluble sugar content and the photosynthetic pigment content in the transgenic Dendrobium officinale. The chloroplasts were well developed when increased, while the knockout protocorms could not develop normally and had no chloroplasts.

Description

The application of RCA gene of variegated orchid and its vector construction method

technical field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to the application of the RCA gene of Spotted orchid and a method for constructing a vector thereof.

Background technique

China is very rich in wild orchids, but all wild orchids are listed as endangered plants, and people's demand for orchids with unique leaf shapes, flower shapes and colors is increasing. After a long-term selection and cultivation of orchids, there have been many variant varieties. According to the different ornamental parts, they are divided into two types: flower art and leaf art. Among them, the leaf spot art orchid originated in China and has gradually been recognized by people in recent years. . The so-called leaf art refers to the relatively stable variation of the characters on the leaves of orchids, including changes in leaf color and leaf shape. The traditional Yeyilan mainly changes the color of the leaves, and most of them are divided into three categories: crystal Yelan, variegated Yelan, and Line Yelan. Among them, Lin Yelan refers to the yellow or white that appears on the green leaves of orchids. Different stripes of different thicknesses and colors can be roughly divided into claw art, edge art, clay art, and medium-through art according to the position and shape of the line distribution. There are many reasons for the formation of leaf art, such as environmental changes, virus invasion, certain nutrient deficiencies, etc. These reasons cause mosaic spots on the leaves, resulting in different "artistic orientations".

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) is abundant in the leaves of plants and plays an important role in regulating phytochrome, carbon assimilation in photosynthesis and photorespiration. In the leaves, the yellow or white stripes of different lengths and thicknesses appear on the leaves, the gene is down-regulated, and existing studies have shown that the expression of Rubisco activity depends on the regulation of RCA, and the role of RCA in RCA Only Rubisco can show the activity of carboxylation or oxygenation. The research and application of the RCA (Rubisco activase) gene that regulates the expression of Rubisco gene in Orchid variegata has become a research direction, and there is no relevant report yet.

SUMMARY OF THE INVENTION

The main purpose of the present invention is to provide a gene fragment that can significantly affect the growth of Dendrobium officinale orchid RCA gene, the nucleotide sequence is shown in SEQ ID NO: 1, the gene fragment is normally expressed in Dendrobium officinale, and promotes the growth of Dendrobium officinale. Growth and development and polysaccharide synthesis of Dendrobium.

Another object of the present invention is to provide a Dendrobium officinale plant model. After knocking out the RCA2 gene, Dendrobium officinale does not form chloroplasts. As a comparative test in the research, this technical method is used for gene editing and new development of orchid plants. Variety breeding provides a reference. The specific RCA2 gene nucleotide sequence is shown in SEQ ID NO: 2, which is a homologous RCA gene obtained from Dendrobium officinale by aligning with the RCA gene sequence of the variegated orchid.

The present invention also provides a recombinant vector of the above-mentioned gene and a construction method of the recombinant vector, comprising the following steps:

(1) Amplification of the RCA gene of Orchid variegata;

The RCA gene sequences obtained in the transcriptome were compared, and it was found that the RCA gene sequence had a 5', and the 3' was amplified by RACE technology to obtain a full-length gene fragment; As a result, the homologous gene RCA2 of RCA in Dendrobium officinale was obtained;

(2) Construction of overexpression vector pCAMBIA1300-35S-RCA and knockout vector pCAMBIA1300DM-OsU6-Cas9;

The primers were designed according to the RCA gene of Orchid variegata, the gene was cloned with a high-fidelity enzyme, and the pCAMBIA1300-35S vector was double-enzyme digested. The full-length sequence and the vector sequence were obtained with complementary sticky ends. The vector fragment was recovered, subjected to in vitro homologous recombination reaction with the PCR product of RCA, transformed into Escherichia coli, picked a single colony for identification, screened out the recombinant by enzyme digestion identification, and constructed a plant-expressed recombinant vector pCAMBIA1300-35S-RCA , and then the recombinant plasmid was obtained;

Two CRISPR target sites were designed according to the cds sequence of the RCA2 gene obtained by homologous alignment, and designed according to the two selected target sites. After ligation, PCR amplification was performed using primers with BsaI restriction sites to obtain a gRNA expression cassette containing the target site, and the amplified target site fragments were recovered and digested with BsaI, and the Cas9 obtained by BsaI restriction was digested with BsaI. The vector was ligated, the component knocked out the vector pCAMBIA1300DM-OsU6-Cas9, the ligated product was electrotransformed into E. coli competent cells, coated with kanamycin-resistant plates for culture, and a single colony was picked for screening of positive clones. Obtain the constructed knockout plasmid.

Further, in the step (2), the two CRISPR target sites are Target 1: ATCCATGGGAGACTCACCGC; Target 2: TGATCAATC AGATGACCAGC; the linker primers of the target sites are Target 1F: GCCGATCCATGG GAGACTCACCGC; Target 1R: AAACGGCG TGAGTCTCCCATGGAT; Target 2F: GTTGTGATCAATCAGATGACCAGC; Target 2R: AAACGCTGGTCATCTG ATTGATCA.

The present invention utilizes the method of RACE to clone the RCA gene from the Orchid variegata, obtains the homologous gene RCA2 of the RCA gene in Dendrobium officinale by using the sequence comparison, and over-expresses and knocks out the expression in Dendrobium officinale, and through Similar to the leaf disc (protocorm) transformation method, it was transformed into wild-type Dendrobium officinale. The experimental results showed that the RCA gene could be expressed normally in the overexpressed transgenic Dendrobium officinale, and the growth of the overexpressed RCA gene was better than that of the control plants. Robust, overexpression of the RCA gene can greatly improve the protein content, soluble sugar content, photosynthetic pigment content, and well-developed chloroplast in transgenic Dendrobium officinale.

Description of drawings:

Figure 1: Gel electrophoresis before and after amplification of RCA gene (A: PCR product of RCA gene 3' end, M: maker2000bp; 3: 3'RACE product B: full-length amplification of RCA gene, M:maker10000bp; 1: RCA gene full length);

Figure 2: Overexpression vector PCR verification gel electrophoresis display;

Figure 3: Diagram showing the construction of gene knockout recombinant vector;

Figure 4: Overexpression and transformation of Dendrobium officinale protocorm (A: infection; B: drying after infection; C: co-culture; D: screening culture; E: proliferation and differentiation culture; F: differentiation and rooting culture);

Figure 5: Knockout transformation of Dendrobium officinale protocorm (A: treatment of protocorm; B: infection of protocorm; C: co-culture; D: transfer into screening culture; E: screening; F: post-screening culture);

Figure 6: PCR detection of transgenic positive plants (A is overexpression, B is knockout, M: DL2000, 1: wild-type plant, 2: positive control, the rest are transgenic plants);

Figure 7: Transgenic and wild-type plant morphology display diagram (A, B, C are transgenic plants; CK is wild plant);

Figure 8: Comparison of photosynthetic pigment content in leaves of wild-type and transgenic Dendrobium officinale plants;

Figure 9: Comparison of soluble protein and sugar content in leaves of wild-type and transgenic Dendrobium officinale plants;

Figure 10: Comparison of chloroplast development in wild type, overexpression and knockout protocorm observed by transmission electron microscope (A, B, C are wild type, D, E, F are overexpression, G, H, I are knockout; Ch, chloroplast; CW, cell wall; M, mitochondria; O, ghrelin; IS, intercellular space; GL, grana lamella; SG, starch granules; N, nucleus).

Detailed ways

In order to be able to understand the technical essence and beneficial effects of the present invention more clearly, the applicant will describe in detail below by way of examples, but the descriptions of the examples are not intended to limit the solution of the present invention. The equivalent transformations that are only formal but not substantial should be regarded as the scope of the technical solutions of the present invention.

In order to avoid excessive unnecessary details, the well-known technologies will not be described in detail in the following embodiments. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The test reagent consumables used in the following examples are conventional biochemical reagents unless otherwise specified; the experimental methods are conventional methods unless otherwise specified.

Example 1

1. Construction of RCA recombinant vectors pCAMBIA1300-35S-RCA and pCAMBIA1300DM-OsU6-Cas9

pCAMBIA1300-35S-RCA is a recombinant plant overexpression vector. The pCAMBIA1300-35S-RCA plant expression vector contains the RCA gene of Orchid variegata. The starting vector is pCAMBIA1301. pCAMBIA1300DM-OsU6-Cas9 is a knockout vector, which contains the RCA2 gene, the homologous gene of the Orchid variegata RCA gene in Dendrobium officinale.

The pCAMBIA1300-35S-RCA plant expression vector obtained by the present invention contains the 35S promoter followed by the RCA gene. pCAMBIA1300DM-OsU6-Cas9 contains promoter U6, and the RCA2 gene, the homologous gene of the RCA gene of Yeyilan in Dendrobium officinale, is used as the target gene for knockout.

The so-called RCA gene is obtained by RACE technology on the basis of the EST sequence in the transcriptome data we constructed. It is named RCA, and the nucleotide sequence is shown in SEQ ID NO: 1. ORF Finder analysis shows that the RCA gene It has a long and complete open reading frame with a total of 1317 bp, the start codon is ATG, and the stop codon is TAG, encoding a total of 541 amino acids. The overall average hydrophilicity of the protein analyzed by ExPASy was -0.285, indicating that the protein was a hydrophilic protein. The RCA2 gene is the nucleotide sequence of the homologous gene of the RCA gene of Yeyilan obtained after the sequence alignment is shown in SEQ ID NO: 2.

2. The recombinant vectors pCAMBIA1300-35S-RCA and pCAMBIA1300DM-OsU6-Cas9 are constructed by the following methods:

(1) Amplification of the RCA gene of Orchid variegata

The RCA gene sequences obtained in our transcriptome were compared, and it was found that the RCA gene sequence had a 5', and the 3' was amplified by RACE technology to obtain the full-length gene sequence.

①PCR primers

Primers were designed based on RCA gene fragments obtained from our transcriptome:

B26: 5'-GACTCTAGACGACATCGATTTTTTTTTTTTTTTTTT-3';

RCA-1: 5'-GTTCAGCTCCCCAGGTCTATACAACA-3';

RCA-2: 5'-AACCCACTTTCTCACTTCATCATCATAC-3'.

② Carry out PCR with the above-mentioned reverse transcription products, the system is as follows: 10xPCR buffer 2.5ul, dNTP mixture (10mM) 2.5ul, RCA1 10.5ul, B26 0.5ul, cDNA 1.5ul, Taq plus 0.25ul and H2O 16.25ul, the total capacity is 25ul;

③The PCR reaction procedure was as follows: pre-denaturation at 96°C for 5 min; denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 2 min, 35 cycles; extension at 72°C for 10 min, termination of the reaction at 4°C.

④Same system and reaction procedure, use the RCR product of the first round as the template to do the second round of PCR, and the primers are RCA-2 and B26.

⑤ The obtained fragment was linked into pMD18-T, and the sequence was determined after restriction enzyme digestion and identification.

⑥ According to the determined sequence and its overlapping region, splicing out the full-length cDNA of the target gene, and designing primers RCA-3: 5'-CAAGGTCCCCCTCATTTTGGGTGTT-3' and RCA-4: 5'-CTCATTTTGGGTGTTTGGGGAGGCA-3' respectively. The full-length sequence was amplified by PCR for further verification. As shown in Figure 1, the target gene fragment is clear and the size is consistent with the expected result.

(2) Construction of RCA gene plant overexpression vector pCAMBIA1300-35S-RCA and knockout vector pCAMBIA1300DM-OsU6-Cas9

Primers were designed according to the RCA gene of Ye Yilan (RCA-BamHIF(5'-3'): TCTGATCAA GAGACAGGATCCATGGCCTCCTCTGTTTCAAC; RCA-SalIR(5'-3'): CATCGGTGCACTAGTGTCGACCTAACGATAGAAAGATCCAG), the gene was cloned with high fidelity enzyme, and BamHI and SalI double enzymes were used. The pCAMBIA1300-35S vector was cut, and the obtained full-length sequence and the vector sequence were brought with complementary sticky ends, the obtained vector fragment was recovered, and the PCR product of RCA was subjected to in vitro homologous recombination reaction, transformed into Escherichia coli, through Recombinant was screened out by restriction enzyme digestion, and the recombinant vector pCAMBIA1300-35S-RCA for plant expression was constructed, and then the recombinant plasmid was obtained. The target gene fragment is clear, the size is consistent with the expected result (1317bp), there is no non-specific amplification band, and the overexpression vector is successfully constructed; Figure 2 shows the overexpression vector diagram.

Two CRISPR target sites (Target1: ATCCAT GGGAGACTCACCGC; Target 2: TGATCAATCAGATGACCAGC) were designed according to the cds sequence of the Dendrobium officinale RCA2 gene obtained by homology alignment, and the linker primers of the target sites were designed according to the two selected target sites:

Target 1F: GCCGATCCATGG GAGACTCACCGC;

Target 1R: AAACGCGGTGAGTCTCCCATGGAT;

Target2F: GTTGTGATCAATCAGATGACCAGC;

Target2R: AAACGCTGGTCATCTG ATTGATCA;

The vector was knocked out by digestion with BsaI, and then ligated with the small fragments annealed to the target site, and then PCR amplification was performed using primers with a BsaI digestion site to obtain a gRNA expression cassette containing the target site. The amplified target site fragment was recovered and digested with BsaI, and then ligated with the Cas9 vector obtained by BsaI digestion. The ligated product was electrotransformed into competent cells of E. coli, and cultured on kanamycin-resistant plates. , pick a single colony to screen for positive clones, and obtain a constructed knockout plasmid. The target gene fragment was clear, the size was consistent with the expected result (432bp), and the knockout vector was successfully constructed; Figure 3 is a schematic diagram of the construction of the knockout vector.

3. Genetic transformation of Agrobacterium and detection of transformants

The competent cells of Agrobacterium were prepared, and the above-constructed plant expression vectors pCAMBIA1300-35S-RCA and pCAMBIA1300DM-OsU6-Cas9 were transferred into Agrobacterium (EHA105) by electric pulse method, and screened on a plate with hygromycin. Transformant colonies. The lysate of Agrobacterium colonies was used as the template of PCR reaction, and the specific primers RCA-BamHIF and RCA-SalIR, Target 2F and Target 2R were used for PCR detection, and the transformant colonies confirmed by colony PCR were used to transform plants.

4. Transforming plants with Agrobacterium containing RCA gene plant overexpression and knockout vectors

A single colony of Agrobacterium carrying plasmids pCAMBIA1300-35S-RCA and pCAMBIA1300DM-OsU6-Cas9 was picked and inoculated into liquid medium for culture, and the cells were collected by centrifugation, and then suspended in MS liquid medium. Infect easily differentiated plant tissues with suspended Agrobacterium, and then obtain transgenic seedlings through tissue culture, and then use antibiotics to screen to obtain transgenic plants. The overexpression genetic transformation process is shown in Figure 4, and the knockout transformation genetic transformation process is shown in Figure 5, and positive results were obtained. Plant or protocorm.

5. Detection of RCA gene insertion in transgenic plants

In order to confirm that the transgenic plants obtained by screening with antibiotics (hygromycin) indeed contain the RCA gene, the screened transgenic plants were further identified by using the 2XT5 Direct PCR Kit (Plant). First, the genomic DNA of the transgenic plants was extracted, and then using the plant genomic DNA as a template, hygromycin-specific primers (HygRF: ACGCGTCGACA TGTCTAAGGGCGAGGAACTC; HygR R: GGACTAGTTTATTTATAGAGTTCGTC CAT) were used for PCR detection to identify whether they were positive plants. The identification results are shown in the figure. 6 shown. 6. Experiments on the changes of morphological and physiological indexes of transgenic plants

The transgenic and control wild-type protocorm were cultivated into seedlings in the medium, and the plant Dendrobium officinale overexpressing the RCA gene grew robustly, carried out the measurement of various physiological indicators, and obtained Figure 7, Table 1, Table 2, Figure 8, The measurement results of Figure 9 and Figure 10, the experimental results show that the RCA gene can be expressed normally in the transgenic overexpressed Dendrobium officinale, and the growth of the transgenic RCA gene is compared to the control plant (non-transgenic wild type), and the transgenic plant is more robust. , overexpression of the RCA gene can greatly increase the photosynthetic pigment content of transgenic Dendrobium officinale, and the chloroplast development is better than that of the wild type, while the knockout protocorm cannot develop into seedlings and there is no chloroplast under the transmission electron microscope. The protein and soluble sugar content of gene plants increased, and the sugar components were detected by GC-MS technology. The results showed that compared with wild-type Dendrobium, the overexpression plants had higher levels of sucrose, arabinose, rhamnose, fucose, and inositol. The contents of , sorbitol, xylitol and maltose increased, indicating that the overexpression of RCA gene affects the glucose metabolism pathway in Dendrobium, plays an important role in the process of photosynthetic carbon assimilation, and promotes the synthesis and accumulation of main carbohydrate metabolites.

Table 1 Detection of positive plant morphological indicators

Table 2 Carbohydrates in transgenic RCA plants

substance Substance class WT RCA Type maltose disaccharide 0.193±0.005a 0.382±0.139a up sucrose disaccharide 19.420±1.990a 20.559±1.486a up trehalose disaccharide 0.656±0.048a 0.491±0.027b down D-arabinose Monosaccharide 0.225±0.056a 0.260±0.030a up D-Fructose Monosaccharide 10.051±1.443a 5.197±0.726b down L-fucose Monosaccharide 0.083±0.005B 0.139±0.008A up D-galactose Monosaccharide 0.536±0.042a 0.499±0.046a down glucose Monosaccharide 28.006±4.401a 17.239±2.809a down Inositol Monosaccharide 1.850±0.212a 2.533±0.541a up L-Rhamnose Monosaccharide 0.309±0.009a 0.310±0.005a up D-Sorbitol Monosaccharide 0.015±0.0001a 0.019±0.004a up Xylitol Monosaccharide 0.012±0.0003a 0.013±0.0002a up

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Claims (1)

1. the application of leaf spot art orchid RCA gene, it is characterized in that, RCA gene can be expressed normally in overexpressing transgenic Dendrobium officinale, and overexpressing RCA gene can increase the content of carbohydrate substance in transgenic Dendrobium officinale, the nucleoside of described RCA gene The acid sequence is shown in SEQ ID NO: 1; the carbohydrate is maltose, sucrose, D-arabinose, L-fucose, inositol, L-rhamnose, D-sorbitol or xylitol.

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