CN116426535B - Cassava MeChlD gene and its silencing system and application - Google Patents
Cassava MeChlD gene and its silencing system and application Download PDFInfo
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- CN116426535B CN116426535B CN202211400304.8A CN202211400304A CN116426535B CN 116426535 B CN116426535 B CN 116426535B CN 202211400304 A CN202211400304 A CN 202211400304A CN 116426535 B CN116426535 B CN 116426535B
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Abstract
The invention provides a cassava MeChlD gene, the nucleotide sequence of which is shown as SEQ ID NO. 1. The invention also provides a silencing fragment selected from cassava MeGRXC gene and application thereof. MeChlD genes are key genes in the photosynthesis process of cassava, and can influence the synthesis of chlorophyll in cassava leaves, so that the net photosynthesis rate of the leaves is influenced. MeChlD gene codes magnesium ion chelating enzyme subunit D, has very high homology with rubber tree HbChlD and castor RcChlD, is up-regulated to express in cassava leaves, is positioned on chloroplasts, can promote the growth of prokaryotic expression recombinant bacteria, and interacts with MeChlM and MePrxQ to participate in photosynthesis. The gene is silenced in cassava by utilizing a gene silencing VIGS technology, so that the total chlorophyll content of cassava plant leaves is obviously reduced, the leaves fade, plant development is bad, the expression of photosynthesis related genes, accumulation of cassava root starch and the like are influenced.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a cassava MeChlD gene, and a silencing system and application thereof.
Background
The cassava (Manihot esculenta Crantz) is a plant of the genus cassava of the family Euphorbiaceae, has rich starch content and wide application, occupies an important role in the food industry, and gradually expands the occupied specific gravity in the energy, chemical and pharmaceutical industries. At present, the demand of people on cassava is changed from the demand quantity to the demand quality, and the higher demand is on the yield and the quality of the cassava. How to cultivate the cassava varieties with higher quality and higher yield is the focus of current cassava research. The method has a certain practical significance in improving photosynthesis of cassava leaves as an entry point and providing theoretical basis for cultivating high-quality and high-yield cassava varieties.
The virus-induced gene silencing system (VIGS) is a genetic technology for inhibiting the expression of plant endogenous genes by inserting recombinant viruses of target gene fragments, and is widely applied to the functional verification of various crop genes at present. Because of long growth cycle of tropical crops, great genetic transformation difficulty and certain difficulty in gene function identification. And the VIGS can rapidly identify the gene function, so that the research of the functional genome of the tropical crops is promoted.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a cassava MeChlD gene, a silencing system and application thereof.
The first aspect of the invention provides a cassava MeChlD gene (hereinafter also referred to as "ChlD", "ChlD gene", etc.), the nucleotide sequence of which is shown in SEQ ID NO. 1.
In a second aspect, the invention provides a protein encoded by the cassava MeChlD gene as described in the first aspect of the invention.
In a third aspect, the invention provides a silencing fragment which is a specific fragment selected from cassava MeGRXC gene, and the nucleotide sequence of the silencing fragment is shown as SEQ ID NO. 2.
In a fourth aspect, the present invention provides a recombinant vector or host bacterium comprising the coding region sequence of the cassava MeChlD gene as described in the first aspect.
As the original vector for constructing the recombinant vector, vectors commonly used in the field of gene recombination, such as viruses, plasmids, and the like, can be used. The invention is not limited in this regard. In one embodiment of the present invention, pCAMBIA1302, pGADT7, pGBKT7, pET-28a, etc. are used as the original vector, but it is understood that other plasmids, viruses, etc. may be used.
In a fifth aspect, the invention provides a recombinant vector or host bacterium comprising the silencing fragment of the third aspect of the invention.
As the original vector for constructing the recombinant vector, vectors commonly used in the field of gene recombination, such as viruses, plasmids, and the like, can be used. The invention is not limited in this regard. In one embodiment of the invention, pCsCMV-NC is used as the original vector, but it should be understood that other plasmids, viruses, etc. may be used.
Preferably, the recombinant vector is pCsCMV-NC recombinant plasmid containing the silencing fragment according to the third aspect of the invention.
In a sixth aspect, the invention provides the use of the cassava MeChlD gene of the first aspect of the invention, or a recombinant vector containing the coding region of the cassava MeChlD gene of the first aspect, in improving the magnesium stress resistance of a prokaryotic expression strain.
In a seventh aspect, the invention provides the use of the cassava MeChlD gene as described in the first aspect of the invention, or the recombinant vector or host bacterium as described in the fourth aspect of the invention, in regulating the growth of cassava plants.
The cassava MeChlD gene in the first aspect of the invention or the recombinant vector or host bacterium in the fourth aspect of the invention is used for transforming cassava, so that cassava plants can be dwarfed and root growth is poor.
In an eighth aspect, the invention provides the use of a silencing fragment according to the third aspect of the invention, or a recombinant vector or host bacterium according to the fifth aspect of the invention, for modulating the chlorophyll content of cassava.
The cassava MeChlD gene in the first aspect of the invention or the recombinant vector or host bacterium in the fourth aspect of the invention is used for transforming cassava, so that the chlorophyll content of the cassava can be reduced.
In a ninth aspect, the invention provides the use of a silencing fragment according to the third aspect of the invention, or a recombinant vector or host bacterium according to the fifth aspect of the invention, for regulating the growth of a cassava plant.
The silent fragment in the third aspect of the invention or the recombinant vector or host bacteria in the fifth aspect of the invention converts cassava, so that the color of young cassava leaves is light green and does not have purple red, the leaves are thinned, the leaves of new leaves fade in a large area, and the leaves are partially or wholly yellowing.
A tenth aspect of the invention provides the use of a silencing fragment according to the third aspect of the invention, or a recombinant vector or host bacterium according to the fifth aspect of the invention, for increasing expression of the RbcL gene (manes.s113700.1), and/or increasing expression of the ChlH gene (manes.08g050600), and/or decreasing expression of the MeChlD gene (manes.14g005700), and/or decreasing expression of the ChlI gene (manes.17g053100), and/or decreasing expression of the ChlM gene (manes.06g126400), and/or decreasing expression of the PEPCase gene (manes.15g093700), and/or decreasing expression of the RbcS gene (manes.13g060800), and/or decreasing expression of the PrxQ gene (manes.16g091900). The disclosed platform of each gene is https:// phytozome-next.
In an eleventh aspect, the invention provides a silencing fragment according to the third aspect of the invention, or a recombinant vector or host bacterium according to the fifth aspect of the invention, for modulating the cassava phenotype, and/or the number of cassava mass, and/or the fresh potato weight, and/or the total starch content of cassava mass.
The silencing fragment described in the third aspect of the invention, or the recombinant vector or host bacterium described in the fifth aspect of the invention, transforms cassava, and after transplanting into a field for 4 months, leaves of the VIGS silencing plant MeChlD-2 still remain flowers and leaves or turn yellow.
The silent fragment of the third aspect of the invention, or the recombinant vector or host bacterium of the fifth aspect of the invention, converts cassava, which can significantly reduce the root number of cassava, the fresh potato weight and the total starch content.
The beneficial effects of the invention are as follows:
1. the MeChlD gene is a key gene in the photosynthesis process of the cassava, and can influence the synthesis of chlorophyll in cassava leaves, so that the photosynthesis rate of the leaves is directly influenced. The coded MeChlD protein contains an ATPase conserved domain (AAA+ATPase domain) and a vWA domain, can be linked with MgPMT protein and PrxQ protein, and participates in the oxidative stress process of plants, thereby influencing the photosynthetic rate of cassava leaves;
2. the MeChlD gene can improve the magnesium stress resistance of the strain;
3. The MeChlD gene can dwarf the cassava plants and cause poor root growth, thereby regulating and controlling the growth of the cassava plants;
4. The silencing fragment selected from MeChlD genes disclosed by the invention is used for converting cassava, so that the total chlorophyll content of cassava plant leaves can be obviously reduced, and the leaves fade and plant dysplasia;
5. The silencing fragment selected from MeChlD genes provided by the invention is used for transforming cassava, so that the expression of RbcL genes and ChlH genes can be improved, and the expression of MeChlD genes, chlI genes, chlM genes, PEPCase genes, rbcS genes, prx-Q genes and the like can be reduced;
6. The silencing segment selected from MeChlD genes provided by the invention is used for transforming cassava, and after being transplanted to a field for 4 months, the VIGS silencing plant MeChlD-2 leaves still retain the phenomenon of flowers and leaves or turn yellow, and meanwhile, the root number of cassava blocks, the weight of fresh potatoes and the total starch content can be obviously reduced, which shows that MeChlD not only participates in photosynthesis (source), but also can influence accumulation (warehouse) of cassava block root starch.
Drawings
FIG. 1 is a phylogenetic tree of CHLD proteins encoded by the cassava MeChlD gene and CHLD proteins of other species.
FIG. 2 shows the results of MeChlD gene tissue-specific expression and subcellular localization assays.
FIG. 3 shows the response of recombinant bacteria pET-28a-ChlD, pET-28a-ChlM, pET-28a-Prx-Q and control bacteria pET-28a to magnesium stress. The asterisks in the figure represent significant differences (P < 0.05) in control bacteria pET-28a under conditions of different concentrations of MgCl 2 stress.
FIG. 4 shows the growth curves of recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrxQ and control bacteria pET-28a under magnesium stress.
FIG. 5 shows the results of drop plate experiments of recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrxQ and control bacteria pET-28a under magnesium ion stress.
FIG. 6A shows the results of normal yeast two-hybrid, and FIG. 6B shows the results of yeast two-hybrid after vector exchange.
FIG. 7 shows the results of the bimolecular fluorescence complementation assay.
FIG. 8 is a phenotypic comparison of normal cassava plants versus transgenic cassava plants.
FIG. 9 is a phenotypic trait of a VIGS-induced gene-silencing plant.
FIG. 10 shows chlorophyll content in VIGS-induced gene silencing plants. Sequentially comprises chlorophyll total content, chlorophyll a content, and chlorophyll b content.
FIG. 11 shows the relative expression levels of MeChlD genes in VIGS-induced gene-silenced plants. pCsCMV-NC is control plant injected with no load, pCsCMV-NC-MeChlD-1 (-2, -3) is experimental plant injected with silencing vector.
FIG. 12 shows the relative expression levels of photosynthetic associated genes in VIGS-induced gene silencing plants. MeChlD-1plant, meChlD-2plant, and MeChlD-3plant were cassava plants injected with silencing vector pCsCMV-NC-MeChlD.
FIG. 13 is a graph showing the characterization of the field phenotype of the VIGS-silenced plant MeChlD-2, the number of tubers, the fresh weight of the potato, and the total starch content of the tubers.
Detailed Description
The invention will be further described with reference to specific embodiments in order to provide a better understanding of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
1. Experimental materials and instrumental reagents
1. Plant material
The extraction material of the total RNA of the cassava leaves is from national trial variety No. 9 (SC 9), and is planted in a Danzhou national cassava germplasm resource garden in 3 months of 2021. The Nicotiana benthamiana (Nictotiana benthamiana) is planted in a constant-temperature culture room in the sea mouth area of the tropical crop variety resource research institute of the national academy of Tropical agriculture, and the cassava variety used in the VIGS experiment is SC9 and is planted in a 3-floor laboratory of the cassava research center of the variety resource research institute of the national academy of Tropical agriculture.
2. Vectors and strains
PEASY-Blunt Zero Cloning Vector was purchased from Beijing full gold biotechnology Co., ltd, subcellular localization vector (over-expression vector) pCAMBIA1302, yeast double hybrid vector pGADT7, pGBKT7 and prokaryotic expression vector pET-28a were all obtained from cassava research center laboratory, and VIGS vector and double molecular fluorescence complementation vector were all purchased from Yita biological Co.
Coli competent TOP10, agrobacterium competent GV3101-pSoup-p19, yeast strain AH109, agrobacterium competent LBA4404 were all purchased from Shanghai Biotechnology Inc., and prokaryotic expression strain BL21 (DE 3) was purchased from Tiangen Biotechnology Inc. (Beijing).
3. Reagents relevant to the experiments
Table 1 main experimental reagents and sources thereof
4. Culture medium and additive
TABLE 2 Medium and composition
If the corresponding liquid culture medium is prepared, the agar powder in the formula is removed. All media formulations in this table require autoclaving at 121 ℃ for 10-20min.
5. Experimental instrument
TABLE 3 Main instrumentation and sources
6. Primer(s)
TABLE 4 primers used in experiments
Note that lowercase letters indicate carrier joints
2. Acquisition of the cassava MeChlD Gene
1. Cassava MeChlD Gene CDS region amplification
And collecting cassava leaves in the root swelling period, and extracting total RNA of the leaves according to specifications by using RNAprep Pure Plant Plus Kit kit of Tiangen company. After quality detection of the total RNA of the extracted leaf, reverse transcription was performed according to the specification using TRANSSCRIPT ONE-Step gDNA Removal AND CDNA SYNTHESIS Super Mix kit (purchased from Beijing full gold biological company). Using reverse transcription product cDNA as amplification template, adopting the following primers:
performing PCR amplification reaction with a PCR reaction system of 2X Max Master Mix (Dye Plus) 25. Mu.L, upstream primer (10. Mu.M) 2. Mu.L, downstream primer (10. Mu.M) 2. Mu.L, template cDNA 2. Mu.L, ddH 2 O to 50. Mu.L. The PCR amplification procedure was 95℃for 5min, 95℃for 30s,55℃for 30s,72℃for 2min, 35 cycles total, and 72℃for 7min.
2. Detection and delivery analysis
The amplified products were detected by gel electrophoresis and recovered using the TIANGEL MIDI Purification Kit purchased from Tiangen corporation. The concentration of the recovered target fragment and the OD260/OD280 value were detected by an ultra-micro spectrophotometer and were used to connect T-vectors (the T-vector used in this experiment was pEASY-Blunt Zero Cloning Vector, purchased from Beijing full gold Biotechnology). 1 mu L T vector was gently mixed with 4. Mu.L of PCR product and reacted at room temperature for 5min. After the reaction, pEASY-Blunt Zero-ChlD vector was obtained. Then transformed directly into E.coli (E.coli competent TOP10, purchased from Shanghai Biometrics only). After picking and shaking, carrying out bacterial liquid PCR identification (PCR program refers to '1 in' obtaining cassava MeChlD genes 'and CDS region amplification of cassava MeChlD genes'), and sending positive bacterial liquid to Guangzhou Ai Ji biological limited company for sequencing, wherein the MeChlD gene sequence is shown as SEQ ID No. 1.
The E.coli bacterial liquid containing recombinant plasmid pEASY-Blunt Zero-MeChlD was inoculated into 7mL of fresh LB liquid medium by sucking 100. Mu.L, and shaking at 37℃and 200rpm for 12-16h. And (3) after the bacterial liquid expansion and shaking are finished, extracting the recombinant plasmid pEASY-Blunt Zero-Me by using a ToloPrep column type plasmid DNA small extraction kit.
3. Analysis of cassava MeChlD Gene bioinformatics
1. Physicochemical Properties of the cassava MeChlD protein
The physicochemical properties of MeChlD proteins were obtained using protParam. The result shows that MeChlD protein is encoded by 754 amino acids, has a molecular weight of about 82.28kDa and has an isoelectric point of 5.81. Wherein 92 of the positive charge residues and 100 of the negative charge residues. From this we have deduced MeChlD that the protein belongs to the class of stable proteins.
The protein MeChlD amino acid sequence was analyzed for hydrophilicity and hydrophobicity using ProtScale software, and the results showed that the protein MeChlD amino acid showed the most significant hydrophobicity for lie, an analysis score of 4.500, and the Arg most significant hydrophilicity, and a score of-4.500. Since the total amino acid sequence is relatively large in the hydrophilic amino acid, it is presumed that MeChlD protein belongs to the hydrophilic protein.
Prabi is used to predict the secondary structure of the encoded protein of the cassava MeChlD gene. The results show that the secondary structure of the protein consists of alpha-helix (39.79%), random coil (38.06%), extended chain region (15.52%), beta-turn (6.63%), and the protein is presumed to be a stable spatial conformation based on the alpha-helix structure.
The result of the three-level structure prediction of MeChlD protein by ExPaSy is consistent with the predicted result of Prabi, namely the space structure of MeChlD protein is still composed of four components, namely beta-corner, random coil, alpha-helix and extension chain.
2. Analysis of cassava MeChlD protein domains and analysis of evolutionary relationship
The homologous sequence of the cassava MeChlD protein is found through a database NCBI, phytozome, uniprot, the highly homologous sequence is found in rice, tobacco, arabidopsis thaliana, citrus, soybean, upland cotton, brazil rubber tree and castor, and the homologous analysis is carried out by using software DNAMAN, so that the proteins all contain similar ATPase conserved domains (AAA+ATPase domain) and vWA domains.
In order to study the phylogenetic relationship between the cassava MeChlD protein and proteins of different species, protein sequences of different species such as cassava (Manihot esculenta), rice (Oryza sativa), tobacco (Nicotiana tabacum), arabidopsis thaliana (Arabidopsis thaliana), citrus (Citrus sinensis), soybean (Glycine soja), upland cotton (Gossypium hirsutum), brazilian rubber tree (H.brasiliensis), castor (Ricinus communication) and the like are selected, and a phylogenetic tree is constructed by using MEGA7.0 software. As shown in FIG. 1, the proteins are mainly divided into two branches, namely tobacco, rice and Arabidopsis thaliana, and one branch is composed of cassava, brazil rubber tree, castor, upland cotton, citrus and soybean, wherein the cassava and Brazil rubber tree in the branches have close evolutionary relationship, and the cassava, the castor are gathered into a subclass.
4. MeChlD Gene tissue specific expression
And obtaining the expression condition of MeChlD genes in tissues such as leaves, axillary buds, tuberous roots, fibrous roots, calli, root tip meristems, stem segments and the like by utilizing a cassava gene expression database. MeChlD genes are highly expressed in leaves, midvein and stem segments.
5. Subcellular localization
The pEASY-Blunt Zero-MeChlD recombinant plasmid is used as a template, and
ChID YXB-F:5'-gagaacacgggggactATGGCCTTCTCTCCAACTA-3' and
ChID YXB-R:5’-aaagttcttctccttt actagATGAACTCTTTAAGGCTGATA-3’
PCR amplification was performed as a primer. PCR reaction was 2×Max Master Mix (Dye Plus) 25. Mu.L, upstream primer (10. Mu.M) 2. Mu.L, downstream primer (10. Mu.M) 2. Mu.L, template cDNA 2. Mu.L, ddH 2 O to 50. Mu.L. The PCR amplification procedure was 95℃for 5min, 95℃for 30s,55℃for 30s,72℃for 2min, 35 cycles total, and 72℃for 7min.
The amplified product is recovered and purified by cutting gel, then a seamless cloning technology is used for constructing a vector, and a ClonExpress II One Step Cloning Kit kit of Vazyme company is selected to connect the PCR product with the pCAMBIA1302 vector to obtain the pCAMBIA1302-ChID-GFP vector. Coli TOP10 competent cells were then transformed and cultured at 37℃for 9-15h. After picking and shaking, carrying out bacterial liquid PCR identification (refer to '2, detection and delivery analysis' in 'obtaining the gene of the cassava MeChlD'), and delivering positive bacterial liquid to the Guangzhou Ai Ji biological limited company for sequencing. And after sequencing, comparing and analyzing, selecting correct positive clone bacterial liquid.
And activating, expanding and shaking positive clone bacterial liquid with correct sequencing, and then extracting plasmids. The correct positive cloning plasmid pCAMBIA1302-MeChlD-GFP was transformed into Agrobacterium LBA4404 competent cells, and pCAMBIA1302-MeChlD-GFP and empty pCAMBIA1302 (control) were transferred into leaf of Nicotiana benthamiana seedlings grown for 28d using transient transformation. After 3d incubation in the constant temperature incubator, observation was performed by using a confocal fluorescence microscope. The results are shown in FIG. 2, and Green Fluorescent Protein (GFP) is concentrated on chloroplasts of the leaf surface cells of the Nicotiana benthamiana, which shows that MeChlD proteins are mainly distributed in the chloroplasts, namely MeChlD proteins are chloroplast proteins.
6. Prokaryotic expression-based magnesium ion stress test analysis
1. Construction of prokaryotic expression vectors
Prokaryotic expression vectors pET-28a-MeChlD, pET-28a-MeChlM and pET-28a-MePrx-Q are respectively constructed by adopting cassava MeChlD gene, meChlM gene and MePrx-Q gene and a vector pET-28a, and the construction method of the prokaryotic expression vectors pET-28a-MeChlD, pET-28a-MeChlM and pET-28a-MePrx-Q is the same as that of a pCAMBIA1302-MeChlD-GFP vector.
2. Transformation of expression Strain BL21
The expression strain BL21 (DE 3) used for transformation is generally stored in a refrigerator at-80 ℃ and purchased from Tiangen corporation, and the specific operation steps are as follows, taking out the competent strain from the refrigerator, and placing the strain in ice for standing and thawing. About 600ng of DNA of the recombinant plasmid was added to the competence, gently mixed by pipetting, and allowed to stand in an ice box for 30min. The sample was placed in a 42 ℃ water bath for heat shock 45s and rapidly cooled in ice for 2min. At the end of cooling, 500. Mu.L of LB liquid medium (without any antibiotics) was added dropwise to the sample, which was placed in a shaker and activated at 37℃for 50min at 200 rpm. 100 mu L of the activated bacterial liquid is uniformly coated on LB solid medium, and the culture is carried out in a 37 ℃ incubator overnight after sealing plates. The monoclonal is picked up by a sterilizing gun head and placed in a centrifuge tube filled with LB liquid medium for culture. 1-2 mu L of the bacterial liquid is taken for PCR identification (refer to ' 2, detection and delivery analysis in ' two, cassava MeChlD genes acquisition '), and the bacterial liquid is stored at-20 ℃ for standby after the identification is successful.
3. Induction of fusion proteins
Recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrx-Q and control bacteria pET-28a bacterial liquid stored in BL21 (DE 3) were removed from the-20 ℃ refrigerator, fresh LB (50. Mu.g/mL Kana) medium was added, and activated at 37 ℃ and 200rpm to OD 600 of 0.6-1.0. IPTG was added to the bacterial liquid and induced at 20℃and 160rpm for 10h.
4. Prokaryotic recombinant bacterium magnesium ion stress condition screening
After the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrx-Q and control bacteria pET-28a are subjected to induced expression, the bacterial solution is centrifuged at 1000rpm at 4 ℃ for 10min, and is resuspended in a fresh LB (50 mug/mL Kana) medium in an ultra clean bench until the OD 600 is 0.6-1.0. The resuspended bacterial solutions were diluted at a volume ratio of 1:100, inoculated into LB (50 ug/mL Kana) medium containing 0, 50, 100, 150 and 200 mmol/L -1MgCl2, cultured at 37℃for 12 hours at 200rpm, and OD600 was measured.
As shown in FIG. 3, the control bacteria and the recombinant bacteria have the same growth vigor after normal culture for 12 hours, and the OD 600 of the control bacteria pET-28a is obviously lower than that of the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM and pET-28a-MePrx-Q after stress for 12 hours at 200 mmol.L - 1MgCl2.
5. Prokaryotic recombinant strain magnesium ion stress growth curve
After the induction of the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrx-Q and control bacteria pET-28a, the recombinant bacteria were inoculated into LB (50. Mu.g/mL Kana) medium containing 200 mmol.L -1MgCl2, cultured at 37 ℃, OD 600 was sampled and measured every 2 hours, and growth curves of the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrx-Q and control bacteria pET-28a were drawn.
As a result, as shown in FIG. 4, the control group pET-28a was significantly higher than the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrx-Q, in which the recombinant bacteria were grown at a growth rate of pET-28a-MeChlD > pET-28a-MeChlM > pET-28a-MePrx-Q (FIG. 4, "control") in the normal LB medium, but the control group pET-28a was significantly lower than the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrx-Q under 200 mmol/L - 1MgCl2 stress conditions, in which the recombinant bacteria were grown at a growth rate of pET-28a-MePrx-Q > pET-28a-MeChlD > pET-28a-MeChlM (FIG. 4, "Experivent").
6. Prokaryotic recombinant strain magnesium ion stress drop plate test analysis
After the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrx-Q and control bacteria pET-28a are subjected to induced expression, the bacterial liquid is centrifuged at 1000rpm for 10 min at 4 ℃, and then resuspended to an OD600 of 0.2 by using fresh LB (50 mug/mL Kana) culture medium, and then subjected to gradient dilution to 100 and 10 -1、10-2、10-3、10-4 times. mu.L of each of the cells was pipetted into LB (50. Mu.g/mL Kana) plate medium containing 200 mmol.L -1MgCl2, and after culturing at 37℃for 12 hours, the growth of recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrx-Q and control bacteria pET-28a were observed.
As shown in FIG. 5, in LB (50. Mu.g/mL Kana) solid medium, recombinant pET-28a-MePrx-Q > control bacteria pET-28a-MeChlD > recombinant pET-28a-MeChlM are sequentially arranged from top to bottom in colony formation number, and when 200 mmol/L -1MgCl2 is added into LB (50. Mu.g/mL Kana) solid medium, recombinant pET-28a-MePrx-Q > recombinant pET-28a-MeChlD > recombinant pET-28a-MeChlM > control bacteria pET-28a are sequentially arranged from top to bottom in colony formation number, which indicates that the magnesium stress resistance of the prokaryotic expression strain is improved by the cassava recombinant protein MeChlD.
7. Yeast double-impurity system
The recombinant vectors pGBKT7-MeChlD, pGBKT7-MeChlM, pGBKT7-MePrx-Q, pGADT7-MeChlD, pGADT7-MeChlM and pGADT7-MePrx-Q are respectively constructed by adopting the cassava MeChlD gene, the MeChlM gene and the MePrx-Q gene together with the vectors pGBKT7 and pGADT7, and the construction method is the same as that of the pCAMBIA1302-MeChlD-GFP vector. And placing the constructed vector at-80 ℃ for standby. The bait vectors pGBKT7/pGADT7, pGBKT7-MeChlD/pGADT7, pGBKT7-MeChlM/pGADT7, pGBKT7-MePrx-Q/pGADT7 and the like are subjected to co-transformation by adopting a PEG/LiAc method. The specific transformation steps are as follows:
Yeast competent cells AH109 were removed from the refrigerator at-80℃and thawed on ice. After complete melting, 2 mug, CARRIER DNA mu L of co-transformed plasmid and 500 mu L of PEG/LiAc are sequentially added into AH109, and after the addition, the mixture is blown and uniformly mixed, and the mixture is placed in a water bath kettle and heated at a constant temperature of 30 ℃ for 30min, and can be overturned and uniformly mixed during the period. The sample was taken out of the 30 ℃ water bath and then placed in a 42 ℃ water bath for 15min at constant temperature. At the end of heating, the sample was placed in a centrifuge, centrifuged at 5000rpm for 30s, the supernatant was discarded, the pellet was resuspended in 400. Mu.L of ddH 2 O, and then centrifuged for 30s to collect the pellet. ddH 2 O50-100. Mu.L was resuspended, and the resuspension was dropped in SD/-Trp-Leu, SD/-Trp-Leu-Ade-His auxotroph solid medium, cultured upside down at 29℃for 48-96h and the plate growth was observed.
The pGBKT7-MeChlD/pGADT7-MeChlM、pGBKT7-MeChlD/pGADT7-MePrx-Q、pGBKT7-MeChlM/pGADT7-MePrx-Q、pGBKT7-MeChlM/pGADT7-MeChlD、pGBKT7-MePrx-Q/pGADT7-MeChlD、pGBKT7-MePrx-Q/pGADT7-MeChlM plasmids were separately co-transformed into yeast cell competent AH109 using the PEG/LiAc method. 100. Mu.L of the heavy suspension is uniformly spread on a plate of SD/-Trp-Leu nutrient selective medium, and the culture is inverted at 29 ℃ for 2-4d, and the colony growth condition is observed. The results show that the combination of pGBKT7/pGADT7, pGBKT7-MeChlD/pGADT7, pGBKT7-MeChlM/pGADT7, pGBKT7-MePrx-Q/pGADT7 can grow normally on SD/-Trp-Leu defect medium plates, which means that bait plasmids pGBKT7-MeChlD, pGBKT7-MeChlM and pGBKT7-MePrx-Q can express normally in yeasts and have no toxic effect on hosts, and the failure to grow normally on SD/-Trp-Leu-Ade-His defect medium plates, which means that the bait plasmids pGBKT7-MeChlD, pGBKT7-MeChlM and pGBKT7-MePrx-Q have no self-activation phenomenon.
6 Monoclonal cells were randomly picked on plates, placed one by one in 15mL centrifuge tubes with 4mL fresh two-phase lacking liquid medium, and shake-cultured at 29℃at 200rpm to slight turbidity. 1mL of the bacterial liquid is sucked up and centrifuged at 12000rpm for 3 s, the supernatant is discarded, 50 mu L of ddH 2 O is used for blowing and mixing uniformly, and the mixture is dripped on a SD/-Trp-Leu-Ade nutrition selective culture medium plate and a SD/-Trp-Leu-Ade-His nutrition selective culture medium plate, and colony growth is observed through 29 ℃ inverted culture. As shown in FIG. 6A, colonies grew on both plates, indicating successful transfer of the vector combination into yeast cells. pGBKT7-MeChlD/pGADT7-MeChlM can grow normally on the three-missing plates, which shows that the cassava genes MeChlD and MeChlM interact, pGBKT7-MeChlD/pGADT7-MePrx-Q can grow normally on the three-missing plates, which shows that the cassava genes MeChlD and MePrx-Q interact, and pGBKT7-MeChlM/pGADT7-MePrx-Q can grow normally on the three-missing plates, which shows that the cassava genes MeChlM and MePrx-Q interact. However, pGBKT7-MeChlD/pGADT7-MeChlM, pGBKT7-MeChlD/pGADT7-MePrx-Q, pGBKT-MeChlM/pGADT 7-MePrx-Q grow aseptically on the four-plate plates, indicating interaction between cassava MeChlD, meChlM, mePrx-Q two by two, but the interaction is not strong, and some intermediate substance may be needed between them. The vector-exchanged combinations pGBKT7-MeChlM/pGADT7-MeChlD, pGBKT7-MePrx-Q/pGADT7-MeChlD, pGBKT7-MePrx-Q/pGADT7-MeChlM were grown similarly in the two-, three-, and four-way gaps (FIG. 6B).
8. Bimolecular fluorescence complementation
The vectors used in this experiment were pNC-BiFC-En, pNC-BiFC-Ecc, pNC-BiFC-Enn-MeChlD, pNC-BiFC-Ecc-MeChlM, pNC-BiFC-Ecc-MePrx-Q. Wherein the pNC-BiFC-En and the pNC-BiFC-Ecc vectors are purchased from Yi field biological company, and the other vectors are constructed by the same person, and the construction method is consistent with that of the VIGS vector pCsCMV-NC-MeChlD. And (3) converting the constructed bimolecular fluorescent vector into agrobacterium GV3101-pSoup-p19, and storing at-80 ℃ for later use after the conversion is successful.
100 Mu L of each of the empty agrobacteria liquid and the recombinant plasmid-containing agrobacteria liquid is taken and added into 700 mu L of liquid YEP culture medium (20 mu g/mL Rif,50 mu g/mL Kana), and the mixture is gently mixed and placed on a 28 ℃ shaker for activation at 200rpm for 6-7h. And placing agrobacterium tumefaciens bacterial solutions containing pNC-BiFC-En, pNC-BiFC-ec, pNC-BiFC-Enn-ChlD, pNC-BiFC-Ecc-ChlM and pNC-BiFC-Ecc-Prx-Q carrier plasmids in a shaking table at 28 ℃, activating at 200rpm, and observing bacterial solution conditions. When the bacterial liquid is slightly turbid, transferring the bacterial liquid into 20mL of fresh YEP liquid culture medium, placing the bacterial liquid in a shaking table, culturing at a constant temperature of 28 ℃, and stopping culturing when the OD 600 is 0.5-1.0. All bacterial solutions were transferred to a 50mL centrifuge tube sterilized at high temperature and placed in a centrifuge for centrifugation at 6000rpm for 10min. Discard the waste liquid and keep the bacterial precipitate. The thalli were washed with 8mL of the now prepared infection solution and placed in a centrifuge for centrifugation at 6000rpm for 10min. Discard the waste liquid and keep the bacterial precipitate. The washing and resuspension were repeated once. (the formulation of the infection liquid is 10mM MESpH 5.8,10mM MgCl 2, 100 mu M AS). Blowing and resuspending the thalli by using a proper amount of aggressive dye liquor. After full resuspension, the OD 600 of the bacterial liquid is regulated by an ultraviolet spectrophotometer, and the optimal range is 0.4-0.7. Mixing the combined bacterial solutions pNC-BiFC-Enn/pNC-BiFC-Ecc、pNC-BiFC-Enn/pNC-BiFC-Ecc-MeChlM、pNC-BiFC-Enn/pNC-BiFC-Ecc-MePrx-Q、pNC-BiFC-Ecc/pNC-BiFC-Enn-MeChlD、pNC-BiFC-Enn-MeChlD/pNC-BiFC-Ecc-MeChlM、pNC-BiFC-Enn-MeChlD/pNC-BiFC-Ecc-MePrx-Q in equal volume respectively, and standing in dark for 3h. Selecting the Nicotiana benthamiana seedlings which are good in growth condition, not flowering and green in leaf blade, and placing under stronger white light 1h before starting injection, so that pores on the surface of the leaf blade are opened, and subsequent infection of injection bacteria liquid is facilitated. And (5) after the bacterial liquid is stood, starting injection. The injection method is consistent with the step of instantaneous injection of the subcellular localization bacterial liquid. After injection, tobacco is dark cultured for 1d, light is used for culturing for 2d, a puncher is used for manufacturing a temporary leaf specimen after the culturing is finished, and the specimen is placed under a laser confocal microscope to observe the fluorescence distribution condition.
The laser confocal results (figure 7) show that in tobacco leaves coexpressing pNC-BiFC-Enn-MeChlD/pNC-BiFC-Ecc-MeChlM and pNC-BiFC-Enn-MeChlD/pNC-BiFC-Ecc-MePrx-Q, obvious yellow fluorescence can be observed through the excitation energy of YEP fluorescence excitation luminescence, and the yellow fluorescence can also coincide with chloroplast autofluorescence (red fluorescence), and orange fluorescence is formed after fusion. Whereas no yellow fluorescence excited by the YEP fluorescence was observed in the tobacco leaves of control pNC-BiFC-Enn/pNC-BiFC-Ecc、pNC-BiFC-Enn/pNC-BiFC-Ecc-MeChlM、pNC-BiFC-Enn/pNC-BiFC-Ecc-MePrx-Q、pNC-BiFC-Ecc/pNC-BiFC-Enn-MeChlD. The above results indicate that cassava gene MeChlD is capable of interacting with MeChlM, mePrx-Q encoded proteins in chloroplasts.
9. Cassava MeChlD Gene overexpression and conversion of cassava
1. Construction of an overexpression vector and transformation of Agrobacterium LBA4404
The construction method of the cassava MeChlD gene over-expression vector pCAMBIA1302-MeChlD is constructed by the same method as that of the subcellular localization vector pCAMBIA 1302-MeChlD-GFP. The constructed vector is transformed into agrobacterium LBA4404, and is placed at-80 ℃ for standby after the transformation is successful.
2. Induction of fragile calli of cassava
(1) Culturing the cassava tissue culture seedlings in a constant temperature culture room at 28 ℃ for 16 hours with a photoperiod until the tissue culture Miao Dingduan reaches the bottle mouth. (2) The cassava tissue culture seedling is cut off with the lateral bud stem segments by about 1cm, the cut stem segments are placed in a CAM (MS+10mg/L6-BA) culture medium, the lateral buds are required to touch the culture medium, and the culture is carried out in the dark at 28 ℃ for 3-4 days. (3) Observing the expansion of lateral buds under a microscope, cutting out the expanded lateral buds, transferring the lateral buds into CIM (MS+12 mg/L picloram) culture medium, and culturing in the dark at 28 ℃ for 16-18 days. (4) The formed embryo and non-embryo are distinguished, the non-embryo is removed, and the embryo is transferred to a new CIM culture medium for continuous culture for 10-14 days. Note that the sector embryo and torpedo embryo are circularly cultured for 3 times at most, and the quality of the callus is affected by excessive culture times. (5) Mature fan-shaped embryos and torpedo embryos are divided into small clusters, transferred to GD culture media, and subjected to dark culture at 28 ℃ for 21 days in 8-12 clusters in each dish of GD culture media to induce the generation of friable calli, namely FEC (Friable embryogenic callus). (6) And selecting the friable callus to a new GD culture medium to keep activity, and using the friable callus in time to avoid the reduction of the friable callus activity caused by overlong culture time. The remaining somatic embryos can be transferred to new GD medium for continued culture to induce new friable calli.
3. Transformation of fragile cassava callus
(1) Mu.L of recombinant Agrobacterium broth was pipetted into 500. Mu.L of fresh YEP broth and shake-activated at 28℃at 200rpm until the broth appeared slightly cloudy. (2) After the activation, 20mL of YEP liquid medium was added for the expansion culture. Culturing at 28deg.C with shaker at 200rpm until the OD 600 of the bacterial liquid is 0.5-1.0. (3) The bacterial liquid is placed in a low-temperature centrifuge, and is centrifuged for 10min at a rotation speed of 5000rpm under the condition of 4 ℃ and the sediment is collected. (4) The cells were washed with GD liquid medium, centrifuged at 5000rpm at 4℃for 10min and collected. Repeating once. (5) The cells were resuspended in GD liquid medium, and 1mL of the resuspended cell OD 600 was aspirated. The value of the resuspended bacteria OD600 was diluted to 0.2 according to the formula A=0.20×20/OD 600[ A means the amount of the resuspended bacteria liquid aspirated, 20mL of which was supplemented with GD (200. Mu.M AS) liquid medium ]. (6) The friable calli were transferred to a sterilized 50mL centrifuge tube, the calli were blown off, centrifuged at 1000rpm for 10min at normal temperature, and transferred to 28℃and shake-infected at 40rpm for 25min. (7) The callus was poured onto absorbent paper covered with a sterile nylon mesh (100 μm), spread out and air dried, taking care that the moisture was sufficiently air dried, which would otherwise affect subsequent growth. (8) Friable calli were transferred together with nylon mesh to GD (200. Mu.M AS) solid medium and co-cultured in the dark at 22℃for 2-3 days. (9) After co-cultivation, the calli were transferred to sterilized 50mL Erlenmeyer flasks, washed 2-3 times with GD (500 mg/L Carb) liquid medium, and the washed calli were still poured onto absorbent paper covered with sterilized nylon mesh (100. Mu.M), flattened and air dried. (10) Friable calli were transferred to GD (250 mg/L Carb) solid medium and photoperiod cultivated at 28℃for 16 hours for 7 days. (11) The calli were moved to GD medium (250 mg/L Carb+5mg/L Hyg) with more antibiotics added and incubated at 28℃for one week with a photoperiod of 16 hours. (12) The callus was moved to a screening medium (MSN+250 mg/L Carb+5mg/L Hyg), incubated at 28℃for one week with a photoperiod of 16 hours. (13) The callus was moved to a higher selection concentration medium (MSN+250 mg/L Carb+8mg/L Hyg), incubated at 28℃for one week with a photoperiod of 16 hours. (14) The calli were then transferred to medium of higher selection concentration (MSN+250 mg/L Carb+15mg/L Hyg), incubated at 28℃for one week with a photoperiod of 16 hours. (15) repeating step (14) until cotyledons are grown. (16) The cotyledon-grown callus was picked and transferred to CEM (100 mg/L Carb+0.1 mg/L6-BA) medium and cultured for 14 days at 28℃for 16 hours. (17) repeating step (16) until the cotyledons root. (18) The rooted cotyledons were transferred to MS medium and photoperiod cultivated at 28℃for 16 hours until seedlings were grown.
4. DNA extraction and PCR detection of transgenic plants
Transgenic tissue culture seedling leaves with good growth vigor are collected, and leaf DNA is extracted by a CTAB method, and 2 XCTAB extracting solution is purchased from Beijing Cool and Leibo technology Co. The DNA of the transgenic cassava plant is used as a template, a pair of new detection primers are formed by hygromycin universal detection primers on a carrier and universal primers on the carrier, PCR detection is carried out, and if the PCR product electrophoresis detection shows that a target band appears and the sequencing result is correct in comparison, the plant is proved to be a transgenic positive plant.
After cotyledons are transferred to an MS culture medium for culture, transgenic cassava plants are observed at intervals of one week, and the leaf morphology, color, rooting condition, plant height and the like of the transgenic cassava plants are recorded by shooting with a camera. As shown in figure 8, after cotyledon cultivation for 8 weeks, the leaves of the transgenic cassava plants are emerald, the plant height is 13cm, the root growth is vigorous, and the leaves of the over-expression transgenic cassava plants are emerald, but the plant height is only 3cm, and the root growth is poor.
10. Analysis of the function of the cassava MeChlD Gene based on the VIGS technology
1. Construction of the VIGS vector pCsCMV-NC-MeChlD
The specific fragment shown in SEQ ID NO.2 (300 bp-554bp of the sequence shown in SEQ ID NO. 1) is selected from the cassava MeChlD gene sequence as a silencing fragment, a primer is designed, and universal joint sequences (expressed in lower case letters) of 20bp NC vectors are added at two ends of the primer, as follows:
MeChlD F:agtggtctctgtccagtcctAACTGCCTTGTTGCTTGG;
MeChlD R: GGTCTCAGCAGACCACAAGTAGACCCAATGAGTCTGTCC. The MeChlD gene is used as a template, and primers MeChID CM-F and MeChID CM-R are used for PCR amplification, and amplification products are recovered. PCR reaction was 2× Max Master Mix (Dye Plus) 25. Mu.L, upstream primer (10. Mu.M) 2. Mu.L, downstream primer (10. Mu.M) 2. Mu.L, template cDNA 2. Mu.L, ddH 2 O to 50. Mu.L. The PCR amplification procedure was 95℃for 5min, 95℃for 30s,55℃for 30s,72℃for 40s for 35 cycles, and 72℃for 7min. The amplified product sequence without carrier linker is shown in SEQ ID NO. 3.
The recovered sequence fragment was ligated with NC system universal expression vector using NC vector seamless cloning method, the ligation system is as shown in Table 5.
TABLE 5 NC clone reaction system
Firstly mixing the PCR recovery product with pCsCMV-NC empty plasmid, adding 5 mu L Mix, blowing 10-20 times by a pipetting gun, fully and uniformly mixing, reacting for 30-60min at 50 ℃, converting 2-5 mu L of reaction product into escherichia coli competent DH 5 alpha, and then picking up a monoclonal to perform overnight culture, bacterial liquid PCR identification and positive clone sequencing. And if the final sequencing result is correct in comparison, the vector construction is completed. The constructed recombinant vector needs to be transformed into GV3101-pSoup-p19 agrobacterium competence for storage.
2. VIGS vector injection plants
The agrobacterium solutions containing pCsCMV-NC-MeChlD recombinant plasmid and pCsCMV-NC empty plasmid are respectively transferred into YEP culture medium containing rifampicin 25 mug/mL and kanamycin 50 mug/mL, placed in a shaking table for shaking and activation at 28 ℃ and 200rpm, and can be transferred into a 50mL triangular flask for expansion culture until OD 600 is 1 when the bacterial solutions are slightly turbid. The bacterial cells were collected by low-speed centrifugation (5000 rpm) at 10 min, and the bacterial solution was resuspended in an aggressive solution (the formulation was consistent with that of tobacco) to an OD 600 of 0.8-1.0. Then inoculating 1mL of injector into plant leaves, inoculating 6-8 mature leaves into each plant, injecting 4-6 parts into each leaf, and injecting 0.5-1mL of bacterial liquid into each leaf. Agrobacterium-inoculated plants containing the empty vector pNC-CsCMV were used as controls. After the injection is completed, the plants are moved to a darkroom for overnight culture, and then are placed outside for normal culture, and the growth condition and the phenotype change of the plants are observed once for 3-4 days. And after obvious morphological changes appear, collecting leaf extract DNA, and carrying out PCR verification to obtain the transgenic positive plant.
3. VIGS plant phenotype detection
And (3) selecting cassava plants with good growth vigor and undamaged leaves for injection inoculation, and observing the growth condition of the plants every 3-4d after infection is completed. The cassava leaf phenotype was found to have changed significantly 3 weeks after inoculation. The leaf morphology of the inoculated plants was photographed with a camera and the results are shown in figure 9, which shows that after 3 weeks of inoculation, the young leaves of the cassava plants inoculated with pCsCMV-NC-MeChlD silencing vector (which germinate after inoculation) turned light green and not purple-red, the leaves were thinned, and the young She Yanse of the cassava plants inoculated with pCsCMV-NC empty vector were light green and had a distinct purple-red color. After 5 weeks of inoculation, young leaves of inoculated plants are gradually matured, and the color difference of the leaves is obvious. The new leaf blades of the cassava plants inoculated with pCsCMV-NC empty vector (developed from young leaves after inoculation) are emerald, the new leaf blades of the cassava plants inoculated with pCsCMV-NC-MeChlD silencing vector are subjected to large-area chlorosis, and the blades are subjected to local or whole yellowing, so that chlorophyll content in the blades can be greatly reduced due to the reduction of the expression quantity of the gene MeChlD.
4. Chlorophyll content determination of VIGS plants
Extracting chlorophyll by acetone-ethanol extraction method. Randomly selecting 3 silent plants, taking 3 mature leaves from each plant, taking 0.2g of the part avoiding the midvein of the leaves, cutting, placing into a 25mL brown volumetric flask, pouring 25mL extracting solution (acetone: ethanol=1:1), and extracting in the dark for 24 hours in a 40 ℃ incubator. The absorbance of the filtrate was measured by an ultraviolet spectrophotometer, and the wavelengths mainly measured were 645nm and 663nm. The following formula is the calculation formula of chlorophyll components:
chlorophyll a (mg/g) = (12.7D663-2.69D645) ×v/W
Chlorophyll b (mg/g) = (22.9D645-4.68D663) ×v/W
Total chlorophyll (mg/g) = (20.21D645+8.02D663) ×v/W
Wherein D663 and D645 are absorbance values of chlorophyll solution at wavelengths of 663nm and 645nm, V is the volume (L) of the extracting solution, and W is the fresh weight (g) of the material.
As shown in FIG. 10, compared with the empty cassava plants (pCsCMV-NC) injected, the contents of chlorophyll a and chlorophyll b in the new leaves of the silencing plants pCsCMV-NC-MeChlD are extremely remarkably reduced (P > 0.001), the ratio of chlorophyll a/b is extremely remarkably increased (P > 0.001), and the total chlorophyll amount is extremely remarkably reduced (P > 0.001).
5. VIGS plant silencing efficiency detection and gene expression quantity identification
The silencing efficiency of gene MeChlD in the VIGS plants is analyzed by a real-time quantitative PCR technology, 0.1-0.2g of new leaves are collected after the VIGS inoculation, total RNA of the leaves is extracted by using a RNAprep Pure Plant Plus Kit kit of Tiangen company, and reverse transcription is carried out by using a TRANSSCRIPT ONE-Step gDNA Removal AND CDNA SYNTHESIS Super Mix kit purchased from Beijing full-scale gold biology company. The expression level of ChlD genes was detected by using a real-time quantitative PCR technique. MeChlD silencing efficiency detection primers are shown in Table 4. Beta. -action was used as an internal reference gene, and quantitative detection was performed using a Real-TIME THERMAL CYCLER fluorescence quantitative instrument from Thermo Fisher Co. The PCR reaction system was 2X ChamQ Universal SYBR QPCR MASTER. Mu.L, 0.4. Mu.L of the upstream primer, 0.4. Mu.L of the downstream primer, 1. Mu.L of the template cDNA and 3.2. Mu.L of DNase-FREE WATER. The PCR amplification procedure was 95℃for 7min, 95℃for 5s,60℃for 30s, 40 cycles total, and 60℃for 1min.
The results (FIG. 11) show that compared with the empty control pCsCMV-NC, the relative expression amount of ChlD gene in cassava plants injected with the silencing vector pCsCMV-NC-MeChlD is 30% -60% of that of the empty control, namely the silencing efficiency is 70% -40%, and the relative expression amount has extremely significant difference (P < 0.001), which indicates that the silencing vector pCsCMV-NC-MeChlD can effectively silence MeChlD gene on cassava.
And detecting the expression quantity of other photosynthetic related genes of cassava, including MeChlM, meChlI, meChlH, mePEPCase, meRbcS, meRbcL, mePrx-Q and the like in plants with the MeChlD genes silenced by a real-time quantitative PCR technology. The detection primers are shown in Table 4, and the detection method is consistent with the silencing efficiency detection method. The results are shown in FIG. 12. In the silent plants, besides MeRbcL, meChlH genes, the expression levels of other genes are in an ascending trend, and the descending amplitude is MEPEPCASE > MeRbcS > MePrx-Q > MeChlM > MeChlI in sequence.
6. VIGS silent plant MeChlD-2 field phenotype identification and determination of root number, fresh weight and total starch content of root tuber
The results (FIG. 13) demonstrate that the VIGS silenced plant MeChlD-2 leaves still retain flowers or turn yellow after 4 months of transplanting into the field. Meanwhile, after the cassava tubers are harvested, the number of tubers, the weight of fresh potatoes and the total starch content in the VIGS silent plant MeChlD-2 are obviously lower than those of a control plant. This illustrates MeChlD not only participating in photosynthesis (source), but also affecting accumulation (pool) of tapioca root starch.
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for this practical use will also occur to those skilled in the art, and are within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
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