Cassava MeChlD gene and its silencing system and application

Abstract

The present invention provides a cassava MeChlD gene, the nucleotide sequence of which is shown in SEQ ID NO: 1. The present invention also provides a silencing fragment selected from the cassava MeGRXC3 gene and its application. The MeChlD gene is a key gene in the photosynthesis process of cassava, which can affect the synthesis of chlorophyll in cassava leaves, and then affect the net photosynthetic rate of the leaves. The MeChlD gene encodes the magnesium ion chelatase subunit D, which has a high homology with the rubber tree HbChlD and the castor RcChlD. It is up-regulated in cassava leaves and located on the chloroplasts. It can promote the growth of prokaryotic expression recombinant bacteria, and interact with MeChlM and MePrxQ to participate in photosynthesis. The gene is silenced in cassava using the gene silencing VIGS technology, which can significantly reduce the total chlorophyll content of cassava plant leaves, cause leaf chlorosis, and cause plant stunting, affecting the expression of photosynthesis-related genes and the accumulation of cassava root starch.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a cassava MeChlD gene and a silencing system and application thereof. Background Art

[0002] Cassava (Manihot esculenta Crantz), a plant of the genus Cassava in the Euphorbiaceae family, is rich in starch and widely used. It not only occupies an important position in the food industry, but also gradually expands its share in the energy, chemical, and pharmaceutical industries. At present, people's demand for cassava has shifted from quantity to quality, and there are higher requirements for cassava yield and quality. How to cultivate higher-quality and high-yield cassava varieties is the focus of current cassava research. Taking improving the photosynthesis of cassava leaves as the starting point, it has certain practical significance to provide a theoretical basis for cultivating high-quality and high-yield cassava varieties.

[0003] Virus-induced gene silencing (VIGS) is a genetic technology that inhibits the expression of endogenous genes in plants by inserting recombinant viruses with target gene fragments. It has been widely used in the verification of gene functions in various crops. Due to the long growth cycle of tropical crops and the difficulty of genetic transformation, there are certain difficulties in the identification of gene functions. VIGS can quickly identify gene functions and promote the research of functional genomics of tropical crops. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a cassava MeChlD gene and a silencing system and application thereof.

[0005] The first aspect of the present invention is to provide a cassava MeChlD gene (hereinafter referred to as "ChlD", "ChlD gene", etc.), the nucleotide sequence of which is shown in SEQ ID NO:1.

[0006] The second aspect of the present invention is to provide a protein encoded by the cassava MeChlD gene according to the first aspect of the present invention.

[0007] The third aspect of the present invention is to provide a silencing fragment, which is a specific fragment selected from the cassava MeGRXC3 gene, and the nucleotide sequence is shown in SEQ ID NO:2.

[0008] The fourth aspect of the present invention is to provide a recombinant vector or host bacteria containing the coding region sequence of the cassava MeChlD gene described in the first aspect of the present invention.

[0009] The original vector for constructing the recombinant vector can adopt the vector commonly used in the field of gene recombination, such as virus, plasmid, etc. The present invention is not limited to this. In a specific embodiment of the present invention, the original vector adopts pCAMBIA1302, pGADT7, pGBKT7, pET-28a, etc., but it should be understood that the present invention can also adopt other plasmids, or viruses, etc.

[0010] The fifth aspect of the present invention is to provide a recombinant vector or host bacteria containing the silencing fragment described in the third aspect of the present invention.

[0011] The original vector for constructing the recombinant vector can be a vector commonly used in the field of gene recombination, such as a virus, a plasmid, etc. The present invention is not limited to this. In a specific embodiment of the present invention, the original vector uses pCsCMV-NC, but it should be understood that the present invention can also use other plasmids, or viruses, etc.

[0012] Preferably, the recombinant vector is a pCsCMV-NC recombinant plasmid containing the silencing fragment described in the third aspect of the present invention.

[0013] The sixth aspect of the present invention is to provide the use of the cassava MeChlD gene described in the first aspect of the present invention, or a recombinant vector containing the coding region of the cassava MeChlD gene described in the first aspect, in improving the ability of prokaryotic expression strains to resist magnesium stress.

[0014] The seventh aspect of the present invention is to provide the use of the cassava MeChlD gene as described in the first aspect of the present invention, or the recombinant vector or host bacteria as described in the fourth aspect of the present invention in regulating the growth of cassava plants.

[0015] The cassava MeChlD gene described in the first aspect of the present invention, or the recombinant vector or host bacteria described in the fourth aspect of the present invention can transform cassava to make the cassava plants dwarfed and the roots grow poorly.

[0016] The eighth aspect of the present invention is to provide the use of the silencing fragment as described in the third aspect of the present invention, or the recombinant vector or host bacteria as described in the fifth aspect of the present invention in regulating the chlorophyll content of cassava.

[0017] The cassava MeChlD gene described in the first aspect of the present invention, or the recombinant vector or host bacteria described in the fourth aspect of the present invention can transform cassava to reduce the chlorophyll content of cassava.

[0018] The ninth aspect of the present invention provides the use of the silencing fragment described in the third aspect of the present invention, or the recombinant vector or host bacteria described in the fifth aspect of the present invention in regulating the growth of cassava plants.

[0019] The silencing fragment described in the third aspect of the present invention, or the recombinant vector or host bacteria described in the fifth aspect of the present invention can transform cassava, so that the color of young cassava leaves becomes light green without purple-red, and the leaves become thinner; the new leaves become largely chlorotic, and the leaves become partially or completely yellow.

[0020] The tenth aspect of the present invention is to provide the use of the silencing fragment described in the third aspect of the present invention, or the recombinant vector or host bacteria described in the fifth aspect of the present invention in increasing the expression of the RbcL gene (Manes.S113700.1), and / or increasing the expression of the ChlH gene (Manes.08G050600), and / or reducing the expression of the MeChlD gene (Manes.14G005700), and / or reducing the expression of the ChlI gene (Manes.17G053100), and / or reducing the expression of the ChlM gene (Manes.06G126400), and / or reducing the expression of the PEPCase gene (Manes.15G093700), and / or reducing the expression of the RbcS gene (Manes.13G060800), and / or reducing the expression of the PrxQ gene (Manes.16G091900). The public platform for the above genes is https: / / phytozome-next.jgi.doe.gov / blast-search.

[0021] The eleventh aspect of the present invention is to provide the silencing fragment described in the third aspect of the present invention, or the recombinant vector or host bacteria described in the fifth aspect of the present invention to regulate cassava phenotype, and / or cassava tuber number, and / or fresh tuber weight, and / or total starch content of cassava tubers.

[0022] The cassava transformed with the silencing fragment described in the third aspect of the present invention, or the recombinant vector or host bacteria described in the fifth aspect of the present invention, was transplanted to the field for 4 months, and the leaves of the VIGS-silenced plant MeChlD-2 still retained the phenomenon of mosaic or yellowing.

[0023] The silencing fragment described in the third aspect of the present invention, or the recombinant vector or host bacteria described in the fifth aspect of the present invention can transform cassava, and significantly reduce the number of cassava tubers, fresh tuber weight and total starch content.

[0024] The beneficial effects of the present invention are as follows:

[0025] 1. The MeChlD gene of the present invention is a key gene in the photosynthesis process of cassava, which can affect the synthesis of chlorophyll in cassava leaves, thereby directly affecting the photosynthetic rate of the leaves. The MeChlD protein encoded by it contains the ATPase conserved domain (AAA+ATPase domain) and the vWA domain, which can be linked with the MgPMT protein and the PrxQ protein to participate in the plant oxidative stress process, thereby affecting the photosynthetic rate of cassava leaves;

[0026] 2. The MeChlD gene of the present invention can improve the strain's ability to resist magnesium stress;

[0027] 3. The MeChlD gene of the present invention can dwarf cassava plants and cause poor root growth, thereby regulating the growth of cassava plants;

[0028] 4. Transforming cassava with the silencing fragment of the MeChlD gene selected from the present invention can significantly reduce the total chlorophyll content of the leaves of cassava plants, causing the leaves to turn green and the plants to develop poorly;

[0029] 5. Transforming cassava with the silent fragment of the MeChlD gene selected from the present invention can increase the expression of the RbcL gene and the ChlH gene, and reduce the expression of the MeChlD gene, the ChlI gene, the ChlM gene, the PEPCase gene, the RbcS gene, the Prx-Q gene, etc.;

[0030] 6. The silencing fragment of the MeChlD gene selected from the present invention was used to transform cassava. Four months after transplanting to the field, the leaves of the VIGS-silenced plant MeChlD-2 still retained the phenomenon of mosaic or yellowing. At the same time, the number of cassava tubers, fresh tuber weight and total starch content were significantly reduced, which shows that MeChlD not only participates in photosynthesis (source), but also affects the accumulation of cassava tuber starch (sink). BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Phylogenetic tree of the CHLD protein encoded by the MeChlD gene in cassava and CHLD proteins from other species.

[0032] Figure 2 The results are the tissue-specific expression and subcellular localization analysis of the MeChlD gene.

[0033] Figure 3 The results of the responses of the recombinant bacteria pET-28a-ChlD, pET-28a-ChlM, pET-28a-Prx-Q and the control bacteria pET-28a to magnesium ion stress. The asterisk in the figure indicates the significant difference (P<0.05) of the control bacteria pET-28a under different concentrations of MgCl2 stress.

[0034] Figure 4 This is the growth curve of the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrxQ and the control bacteria pET-28a under magnesium ion stress.

[0035] Figure 5These are the results of the drop plate test of the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrxQ and the control bacteria pET-28a under magnesium ion stress conditions.

[0036] Figure 6 A is the normal yeast two-hybrid result; Figure 6 B is the yeast two-hybridization result after vector exchange.

[0037] Figure 7 Analyze the results of bimolecular fluorescence complementation experiments.

[0038] Figure 8 Comparison of the phenotypes of normal cassava plants and transgenic cassava plants.

[0039] Fig. 9 These are the phenotypic traits of VIGS-induced gene silencing plants.

[0040] Fig.10 The chlorophyll content in the VIGS-induced gene silencing plants is shown in the following order: total chlorophyll content; chlorophyll a content; and chlorophyll b content.

[0041] Fig.11 The relative expression of MeChlD gene in VIGS-induced gene silencing plants. pCsCMV-NC is the control plant injected with empty vector; pCsCMV-NC-MeChlD-1 (-2, -3) is the experimental group plant injected with silencing vector.

[0042] Fig.12 Relative expression levels of photosynthesis-related genes in VIGS-induced gene silencing plants. MeChlD-1plant, MeChlD-2plant, and MeChlD-3plant are cassava plants injected with the silencing vector pCsCMV-NC-MeChlD.

[0043] Fig.13 To identify the field phenotype of the VIGS silenced plant MeChlD-2 and determine the number of tubers, fresh weight of tubers and total starch content of tubers. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to the accompanying drawings in conjunction with specific embodiments to better understand the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Where the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be obtained commercially.

[0045] 1. Experimental Materials and Instruments

[0046] 1. Plant materials

[0047] The extraction material of total RNA from cassava leaves came from the national approved variety South China No. 9 (SC9), which was planted in the Danzhou National Cassava Germplasm Resource Garden in March 2021. Nicotiana benthamiana was planted in the constant temperature culture room of the Haikou Branch of the Institute of Tropical Crops Resources, Chinese Academy of Tropical Agricultural Sciences; the cassava variety used in the VIGS experiment was SC9, which was planted in the laboratory on the 3rd floor of the Cassava Research Center, Institute of Tropical Crops Resources, Chinese Academy of Tropical Agricultural Sciences.

[0048] 2. Vectors and strains

[0049] pEASY-Blunt Zero Cloning Vector was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; subcellular localization vector (overexpression vector) pCAMBIA1302, yeast two-hybrid vector pGADT7, pGBKT7 and prokaryotic expression vector pET-28a were all from the Cassava Research Center Laboratory; VIGS vector and bimolecular fluorescence complementation vector were purchased from Yitian Biotechnology Co., Ltd.

[0050] Escherichia coli competent TOP10, Agrobacterium competent GV3101-pSoup-p19, yeast strain AH109, ​​and Agrobacterium competent LBA4404 were all purchased from Shanghai Weidi Biotechnology Co., Ltd.; the prokaryotic expression strain BL21 (DE3) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0051] 3. Reagents used in the experiment

[0052] Table 1 Main experimental reagents and their sources

[0053]

[0054] 4. Culture medium and additives

[0055] Table 2 Culture medium and composition

[0056]

[0057]

[0058] If you are preparing the corresponding liquid culture medium, just delete the agar powder component in the formula. All the culture media in this table need to be sterilized by high pressure at 121℃ for 10-20min.

[0059] 5. Experimental instruments

[0060] Table 3 Main instruments and equipment and their sources

[0061]

[0062] 6. Primers

[0063] Table 4 Primers used in the experiment

[0064]

[0065]

[0066] Note: Lowercase letters represent vector adapters

[0067] 2. Acquisition of MeChlD gene from cassava

[0068] 1. Amplification of the CDS region of the MeChlD gene in cassava

[0069] Cassava leaves were collected during the root expansion period, and the RNAprep Pure Plant Plus Kit from Tiangen was used to extract total RNA from the leaves according to the instructions. After the extracted total RNA from the leaves passed the quality inspection, it was reverse transcribed using the TransScript One-Step gDNA Removal and cDNA Synthesis Super Mix Kit (purchased from Beijing Quanshijin Biological Company) according to the instructions. The reverse transcription product cDNA was used as the amplification template, and the following primers were used:

[0070]

[0071] Carry out PCR amplification reaction, the PCR reaction system is: 2× Max Master Mix (Dye Plus) 25 μL, upstream primer (10 uM) 2 μL, downstream primer (10 uM) 2 μL, template cDNA 2 μL, ddH2O to 50 μL. PCR amplification program: 95℃5min; 95℃30s, 55℃30s, 72℃2min, 35 cycles; 72℃7min.

[0072] 2. Testing and analysis

[0073] The amplified product was detected by gel electrophoresis, and the product was recovered using the TIANgel MidiPurification Kit purchased from Tiangen Company. The concentration and OD260 / OD280 value of the recovered target fragment were detected by an ultra-micro spectrophotometer. If it met the standard, it could be used to connect to the T vector (the T vector used in this experiment was pEASY-BluntZero Cloning Vector, purchased from Beijing Quanshijin Biological Company). 1 μL of T vector was gently mixed with 4 μL of PCR product and reacted at room temperature for 5 minutes. After the reaction, the pEASY-Blunt Zero-ChlD vector was obtained. Then it was directly transformed into Escherichia coli (the competent Escherichia coli was TOP10, purchased from Shanghai Weidi Biological Company). After picking the bacteria and shaking the bacteria, the bacterial solution was identified by PCR (the PCR program refers to "1. Amplification of the CDS region of the cassava MeChlD gene" in "II. Acquisition of the cassava MeChlD gene"). The positive bacterial solution was sent to Guangzhou Aiji Biological Co., Ltd. for sequencing. The MeChlD gene sequence is shown in SEQ ID No. 1.

[0074] 100 μL of E. coli containing the recombinant plasmid pEASY-Blunt Zero-MeChlD was inoculated into 7 mL of fresh LB liquid medium and cultured at 37°C with a shaker at 200 rpm for 12-16 hours. After the bacterial solution was amplified, the recombinant plasmid pEASY-Blunt Zero-Me was extracted using the ToloPrep column-based plasmid DNA small-scale extraction kit.

[0075] 3. Bioinformatics analysis of cassava MeChlD gene

[0076] 1. Physicochemical properties of cassava MeChlD protein

[0077] ProtParam was used to obtain the physicochemical properties of MeChlD protein. The results showed that MeChlD protein was encoded by 754 amino acids, with a molecular weight of approximately 82.28 kDa and an isoelectric point of 5.81. Among them, there were 92 positively charged residues and 100 negatively charged residues. Therefore, we concluded that MeChlD protein is a stable protein.

[0078] The hydrophilicity and hydrophobicity of the amino acid sequence of MeChlD protein were analyzed using ProtScale software. The results showed that among the amino acids in MeChlD protein, Ile had the most significant hydrophobicity, with an analysis score of 4.500; Arg had the most significant hydrophilicity, with a score of -4.500. In the total amino acid sequence, hydrophilic amino acids accounted for a large proportion, so it can be inferred that MeChlD protein is a hydrophilic protein.

[0079] Prabi was used to predict the secondary structure of the protein encoded by the cassava MeChlD gene. The results showed that the secondary structure of the protein was composed of ɑ-helix (39.79%), random coil (38.06%), extended chain region (15.52%), and β-turn (6.63%), which indicates that the protein is a stable spatial conformation based on the ɑ-helix structure.

[0080] The tertiary structure of MeChlD protein was predicted using ExPaSy, and the results were consistent with those predicted by Prabi, that is, the spatial structure of MeChlD protein is still composed of four major components, namely β-turn, random coil, α-helix, and extended chain.

[0081] 2. Analysis of the structural domain and evolutionary relationship of cassava MeChlD protein

[0082] Homologous sequences of cassava MeChlD protein were searched through the databases NCBI, Phytozome, and Uniprot, and highly homologous sequences were found in rice, tobacco, Arabidopsis, citrus, soybean, upland cotton, Brazilian rubber tree, and castor. Homology analysis was performed using the software DNAMAN, and it was found that these proteins all contain similar ATPase conserved domains (AAA+ATPase domain) and vWA domains.

[0083] In order to study the phylogenetic relationship between cassava MeChlD protein and proteins from different species, protein sequences from different species such as cassava (Manihot esculenta), rice (Oryza sativa), tobacco (Nicotiana tabacum), Arabidopsis thaliana, citrus (Citrus sinensis), soybean (Glycine soja), upland cotton (Gossypium hirsutum), Brazilian rubber tree (H. brasiliensis), and castor (Ricinus communis) were selected, and a phylogenetic tree was constructed using MEGA7.0 software. The results are shown in Figure 1 As shown, these proteins are mainly divided into two branches: tobacco, rice, and Arabidopsis are one branch; cassava, rubber tree, castor, upland cotton, citrus, and soybean are another branch. In this branch, cassava, rubber tree, and castor have a close evolutionary relationship and are clustered into one subclass.

[0084] 4. Tissue-specific expression of MeChlD gene

[0085] Using the cassava gene expression database, the expression of MeChlD gene in leaves, axillary buds, tuberous roots, fibrous roots, callus, root apical meristem, stem apical meristem, stem segments, etc. MeChlD gene is highly expressed in leaves, midribs and stem segments.

[0086] 5. Subcellular localization

[0087] Using pEASY-Blunt Zero-MeChlD recombinant plasmid as template,

[0088] ChID YXB-F: 5'-gagaacacgggggactATGGCCTTCTCTCCAACTA-3' and

[0089] ChID YXB-R: 5'-aaagttcttctccttt actagATGAACTCTTTAAGGCTGATA-3'

[0090] As primers, PCR amplification was performed. The PCR reaction system was 2× Max Master Mix (Dye Plus) 25 μL, upstream primer (10 μM) 2 μL, downstream primer (10 μM) 2 μL, template cDNA 2 μL, ddH2O to 50 μL. PCR amplification program: 95℃5min; 95℃30s, 55℃30s, 72℃2min, 35 cycles; 72℃7min.

[0091] After gel excision, the amplified product was purified and then constructed using seamless cloning technology. The PCR product was connected to the pCAMBIA1302 vector using Vazyme's ClonExpress II One Step Cloning Kit to obtain the pCAMBIA1302-ChID-GFP vector. Then the competent cells of E. coli TOP10 were transformed and cultured at 37°C for 9-15h. After the bacteria were picked and shaken, the bacterial solution was identified by PCR (refer to "2. Detection and analysis" in "2. Acquisition of cassava MeChlD gene"), and the positive bacterial solution was sent to Guangzhou Aiji Biotechnology Co., Ltd. for sequencing. After sequencing comparison analysis, the correct positive clone bacterial solution was selected.

[0092] The positive cloned bacterial solution with correct sequencing was activated and propagated, and then the plasmid was extracted. The correct positive cloned plasmid pCAMBIA1302-MeChlD-GFP was transformed into Agrobacterium LBA4404 competent cells, and pCAMBIA1302-MeChlD-GFP and empty pCAMBIA1302 (control) were transferred into the leaves of Nicotiana benthamiana seedlings planted for 28 days using the transient transformation method. After 3 days of cultivation in a constant temperature culture room, the leaves were observed under a confocal fluorescence microscope. The results are shown in the figure. Figure 2As shown, green fluorescent protein (GFP) is concentrated in the chloroplasts of epidermal cells of Nicotiana benthamiana leaves, indicating that the MeChlD protein is mainly distributed in the chloroplasts, that is, the MeChlD protein is a chloroplast protein.

[0093] VI. Analysis of magnesium ion stress test based on prokaryotic expression

[0094] 1. Construction of prokaryotic expression vector

[0095] Cassava MeChlD gene, MeChlM gene and MePrx-Q gene were used to construct prokaryotic expression vectors pET-28a-MeChlD, pET-28a-MeChlM and pET-28a-MePrx-Q respectively. The construction method of prokaryotic expression vectors pET-28a-MeChlD, pET-28a-MeChlM and pET-28a-MePrx-Q was the same as that of pCAMBIA1302-MeChlD-GFP vector.

[0096] 2. Transformation of expression strain BL21

[0097] The expression strain BL21 (DE3) used for transformation is generally stored in a -80℃ refrigerator and purchased from Tiangen Company. The specific operation steps are as follows: Take out the competent state from the refrigerator and let it stand in ice to melt. Add about 600ng of recombinant plasmid DNA to the competent state, gently blow and mix with a pipette, and let it stand in an ice box for 30min. Heat shock the sample in a 42℃ water bath for 45s, and quickly insert the sample into ice to cool for 2min. After cooling, add 500μL of LB liquid culture medium (without any antibiotics) to the sample, place it in a shaker, and activate it at 37℃, 200rpm for 50min. Take 100μL of activated bacterial solution and evenly spread it on the LB solid culture medium. After sealing the plate, invert it in a 37℃ incubator for overnight culture. Pick up a single clone with a sterilized pipette tip and culture it separately in a centrifuge tube containing LB liquid culture medium. Take 1-2 μL of the bacterial solution for PCR identification (refer to "2. Detection and analysis" in "2. Acquisition of cassava MeChlD gene"). After successful identification, store the bacterial solution at -20°C for future use.

[0098] 3. Induction of fusion protein

[0099] Take out the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrx-Q, and control bacteria pET-28a stored in BL21 (DE3) from the -20°C refrigerator, add fresh LB (50 μg / mL Kana) medium, and activate at 37°C, 200 rpm until OD 600IPTG was added to the bacterial solution and induced at 20°C and 160 rpm for 10 h.

[0100] 4. Screening of magnesium ion stress conditions for prokaryotic recombinant bacteria

[0101] After the induction of expression of the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrx-Q and the control bacteria pET-28a, the bacterial solution was centrifuged at 1000 rpm for 10 min at 4 °C and resuspended in fresh LB (50 μg / mL Kana) medium in a clean bench to an OD of 600 The resuspended bacterial solution was diluted at a volume ratio of 1:100 and then inoculated into 0, 50, 100, 150, and 200 mmol·L -1 The cells were cultured in LB (50 ug / mL Kana) medium containing MgCl2 at 37°C and 200 rpm for 12 h, and the OD600 value was measured.

[0102] The results are as follows Figure 3 As shown, after 12 h of normal culture, the growth of the control bacteria and the recombinant bacteria was the same; and at 200 mmol·L - 1 OD of control bacteria pET-28a after MgCl2 stress for 12 h 600 They were significantly lower than the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, and pET-28a-MePrx-Q.

[0103] 5. Growth curve of prokaryotic recombinant bacteria under magnesium ion stress

[0104] After the induction of expression of the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrx-Q and the control bacteria pET-28a, the bacterial solution was dropped into a solution containing 200 mmol·L -1 The cells were cultured in LB medium (50 μg / mL Kana) containing MgCl2 at 37°C and the OD was measured every 2 hours. 600 , and draw the growth curves of the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrx-Q and the control bacteria pET-28a.

[0105] The results are as follows Figure 4As shown: After culturing for 12 hours under normal LB medium conditions, the growth rate of the control group pET-28a was significantly higher than that of the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, and pET-28a-MePrx-Q, among which the growth rate of the recombinant bacteria was pET-28a-MeChlD>pET-28a-MeChlM>pET-28a-MePrx-Q( Figure 4 "Control"); but at 200mmol·L - 1 Under MgCl2 stress conditions, the control bacteria pET-28a was significantly lower than the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, and pET-28a-MePrx-Q, and the growth rate of the recombinant bacteria was pET-28a-MePrx-Q>pET-28a-MeChlD>pET-28a-MeChlM( Figure 4 “Experiment”).

[0106] 6. Analysis of magnesium ion stress drop plate test of prokaryotic recombinant bacteria

[0107] After the induction of expression of the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrx-Q and the control bacteria pET-28a, the bacterial solution was centrifuged at 1000 rpm for 10 min at 4 °C, and then resuspended in fresh LB (50 μg / mL Kana) medium to an OD600 of 0.2, and then graded diluted to 100, 10 -1 , 10 -2 , 10 -3 , 10 -4 Pipette 5 μL of bacterial solution onto a drop of 200 mmol·L -1 The growth of the recombinant bacteria pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrx-Q and the control bacteria pET-28a was observed after culturing in LB (50 μg / mL Kana) plate medium containing MgCl2 at 37°C for 12 h.

[0108] The results are as follows Figure 5 As shown in the figure: In LB (50μg / mL Kana) solid medium, the colony formation number is from high to low: recombinant bacteria pET-28a-MePrx-Q> control bacteria pET-28a> recombinant bacteria pET-28a-MeChlD> recombinant bacteria pET-28a-MeChlM; when 200mmol·L Kana was added to LB (50μg / mL Kana) solid medium, the colony formation number is from high to low: recombinant bacteria pET-28a-MePrx-Q> control bacteria pET-28a> recombinant bacteria pET-28a-MeChlD> recombinant bacteria pET-28a-MeChlM -1When MgCl2 was added, the number of colony formation was as follows: recombinant bacteria pET-28a-MePrx-Q>recombinant bacteria pET-28a-MeChlD>recombinant bacteria pET-28a-MeChlM>control bacteria pET-28a, indicating that the cassava recombinant protein MeChlD improved the ability of prokaryotic expression strains to resist magnesium stress.

[0109] VII. Yeast Two-hybrid System

[0110] Cassava MeChlD gene, MeChlM gene, MePrx-Q gene were used to construct recombinant vectors pGBKT7-MeChlD, pGBKT7-MeChlM, pGBKT7-MePrx-Q, pGADT7-MeChlD, pGADT7-MeChlM, pGADT7-MePrx-Q with vectors pGBKT7 and pGADT7, respectively. The construction method was the same as that of pCAMBIA1302-MeChlD-GFP vector. The constructed vectors were stored at -80 °C for later use. The bait vectors pGBKT7 / pGADT7, pGBKT7-MeChlD / pGADT7, pGBKT7-MeChlM / pGADT7, pGBKT7-MePrx-Q / pGADT7 and other combinations were co-transformed using the PEG / LiAc method. The specific transformation steps are as follows:

[0111] Take out the yeast competent cell AH109 from the -80℃ refrigerator and place it on ice to melt. After complete melting, add 2μg of the co-transformed plasmid, 10μL of Carrier DNA and 500μL of PEG / LiAc to AH109 in sequence, mix by blowing after adding, and place it in a water bath at 30℃ for 30min, during which it can be turned over and mixed. Take out the sample from the 30℃ water bath and place it in a 42℃ water bath for 15min. After heating, place the sample in a centrifuge, centrifuge at 5000rpm for 30s, discard the supernatant, resuspend the precipitate with 400μL of ddH2O, and then centrifuge for 30s to collect the precipitate. Resuspend in 50-100 μL ddH2O, drop the resuspended liquid into SD / -Trp-Leu, SD / -Trp-Leu-Ade-His nutrient-deficient solid culture medium, culture upside down at 29°C for 48-96 hours and observe the growth on the plate.

[0112] The pGBKT7-MeChlD / pGADT7-MeChlM, pGBKT7-MeChlD / pGADT7-MeChlM, pGBKT7-MeChlD / pGADT7-MePrx-Q, pGBKT7-MeChlM / pGADT7-MePrx-Q, pGBKT7-MeChlM / pGADT7-MeChlD, pGBKT7-MePrx-Q / pGADT7-MeChlD, and pGBKT7-MePrx-Q / pGADT7-MeChlM plasmids were co-transformed into yeast cell competent AH109 by PEG / LiAc method. 100 μL of the resuspension was evenly spread on SD / -Trp-Leu nutrient selective medium plates, inverted and cultured at 29°C for 2-4 days, and the growth of colonies was observed. The results showed that the combinations of pGBKT7 / pGADT7, pGBKT7-MeChlD / pGADT7, pGBKT7-MeChlM / pGADT7, and pGBKT7-MePrx-Q / pGADT7 could all grow normally on SD / -Trp-Leu defective culture medium plates, indicating that the bait plasmids pGBKT7-MeChlD, pGBKT7-MeChlM, and pGBKT7-MePrx-Q could be expressed normally in yeast and had no toxic effects on the host; the inability to grow normally on SD / -Trp-Leu-Ade-His defective culture medium plates indicated that the bait plasmids pGBKT7-MeChlD, pGBKT7-MeChlM, and pGBKT7-MePrx-Q had no self-activation phenomenon.

[0113] Randomly pick 6 single clones on the plate, place them one by one in a 15mL centrifuge tube filled with 4mL fresh dilution medium, and culture at 29℃, 200rpm shaking until slightly turbid. Pipette 1mL of bacterial solution and centrifuge at 12000rpm for 30s, discard the supernatant, mix with 50μL ddH2O, drop on SD / -Trp-Leu-Ade nutrient selective medium plate and SD / -Trp-Leu-Ade-His nutrient selective medium plate, and invert and culture at 29℃ to observe the growth of colonies. Figure 6As shown in A, colonies grew on both the two-deficiency plates, indicating that the vector combination has been successfully transferred into yeast cells. pGBKT7-MeChlD / pGADT7-MeChlM can grow normally on the three-deficiency plates, indicating that the cassava genes MeChlD and MeChlM interact with each other; pGBKT7-MeChlD / pGADT7-MePrx-Q can grow normally on the three-deficiency plates, indicating that the cassava genes MeChlD and MePrx-Q interact with each other; pGBKT7-MeChlM / pGADT7-MePrx-Q can grow normally on the three-deficiency plates, indicating that the cassava genes MeChlM and MePrx-Q interact with each other. However, pGBKT7-MeChlD / pGADT7-MeChlM, pGBKT7-MeChlD / pGADT7-MePrx-Q, and pGBKT7-MeChlM / pGADT7-MePrx-Q had no colony growth on the four-deficiency plate, indicating that there was interaction between cassava MeChlD, MeChlM, and MePrx-Q, but the interaction was not strong, and an intermediate substance may be needed to mediate between them. The growth of the vector-exchange combinations pGBKT7-MeChlM / pGADT7-MeChlD, pGBKT7-MePrx-Q / pGADT7-MeChlD, and pGBKT7-MePrx-Q / pGADT7-MeChlM in two-deficiency, three-deficiency, and four-deficiency was similar ( Figure 6 B).

[0114] 8. Bimolecular Fluorescence Complementation

[0115] The vectors used in this experiment are pNC-BiFC-Enn, pNC-BiFC-Ecc, pNC-BiFC-Enn-MeChlD, pNC-BiFC-Ecc-MeChlM, and pNC-BiFC-Ecc-MePrx-Q. Among them, pNC-BiFC-Enn and pNC-BiFC-Ecc vectors were purchased from Yitian Biological Company, and the other vectors were constructed by myself. The construction method is consistent with the construction of VIGS vector pCsCMV-NC-MeChlD. The constructed bimolecular fluorescent vector was transformed into Agrobacterium GV3101-pSoup-p19 and stored at -80℃ for future use after successful transformation.

[0116] Take 100 μL of the Agrobacterium culture containing empty vector and the Agrobacterium culture containing recombinant plasmid, add them to 700 μL of liquid YEP medium (20 μg / mL Rif, 50 μg / mL Kana), mix gently and place in a 28°C shaker, activate at 200 rpm for 6-7 hours. Place the Agrobacterium culture containing pNC-BiFC-Enn, pNC-BiFC-Ecc, pNC-BiFC-Enn-ChlD, pNC-BiFC-Ecc-ChlM, pNC-BiFC-Ecc-Prx-Q vector plasmids in a 28°C shaker, activate at 200 rpm, and observe the culture. When the culture becomes slightly turbid, transfer it to 20 mL of fresh YEP liquid culture medium, place it in a shaker, and culture at a constant temperature of 28°C. When OD 600 Stop culturing when it is 0.5-1.0. Transfer all the bacterial liquid to a high-temperature sterilized 50mL centrifuge tube and centrifuge it at 6000rpm for 10 minutes. Discard the waste liquid and keep the bacterial pellet. Wash the bacteria with 8mL of freshly prepared infection solution and centrifuge it at 6000rpm for 10 minutes. Discard the waste liquid and keep the bacterial pellet. Repeat the washing and resuspending once. (The formula of the infection solution is: 10mM MESpH 5.8, 10mM MgCl2, 100μM AS). Use an appropriate amount of infection solution to blow and resuspend the bacteria. After sufficient resuspension, use a UV spectrophotometer to adjust the OD of the bacterial liquid. 600 , the optimal range is 0.4-0.7. Mix 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 volumes and let stand in the dark for 3 hours. Select Nicotiana benthamiana seedlings with good growth, no flowering and green leaves, and place them under strong white light 1 hour before injection to open the stomata on the leaf surface, which is convenient for infection of the subsequent injection of bacterial solution. Injection can be started after the bacterial solution has been left to stand. The injection method was consistent with the above-mentioned subcellular localization bacterial solution instantaneous injection steps. After the injection, the tobacco was cultured in the dark for 1 day and in the light for 2 days. After the culture was completed, a temporary leaf specimen was made using a puncher and the specimen was placed under a laser confocal microscope to observe the fluorescence distribution.

[0117] Laser confocal microscopy results ( Figure 7) showed that in tobacco leaves co-expressing pNC-BiFC-Enn-MeChlD / pNC-BiFC-Ecc-MeChlM and pNC-BiFC-Enn-MeChlD / pNC-BiFC-Ecc-MePrx-Q, obvious yellow fluorescence could be observed after excitation of YEP fluorescence, and the yellow fluorescence could also overlap with the chloroplast autofluorescence (red fluorescence), forming orange fluorescence after fusion. However, no yellow fluorescence excited by YEP fluorescence was observed in tobacco leaves of the control groups pNC-BiFC-Enn / pNC-BiFC-Ecc, pNC-BiFC-Enn / pNC-BiFC-Ecc-MeChlM, pNC-BiFC-Enn / pNC-BiFC-Ecc-MePrx-Q, and pNC-BiFC-Ecc / pNC-BiFC-Enn-MeChlD. The above results indicate that the proteins encoded by the cassava gene MeChlD, MeChlM, and MePrx-Q can interact in chloroplasts.

[0118] IX. Cassava MeChlD gene overexpression and transformation of cassava

[0119] 1. Construction of overexpression vector and transformation of Agrobacterium tumefaciens LBA4404

[0120] The construction method of cassava MeChlD gene overexpression vector pCAMBIA1302-MeChlD is the same as that of subcellular localization vector pCAMBIA1302-MeChlD-GFP vector. The constructed vector was transformed into Agrobacterium LBA4404 and stored at -80°C for future use.

[0121] 2. Induction of brittle callus in cassava

[0122] (1) Place the cassava tissue culture seedlings in a constant temperature culture room at 28°C and a 16-hour photoperiod until the top of the tissue culture seedlings reaches the bottle mouth. (2) Cut off about 1 cm of the stem segment with lateral buds from the cassava tissue culture seedlings, place the cut stem segment in CAM (MS+10mg / L6-BA) medium, and make sure that the lateral buds touch the medium. Culture in the dark at 28°C for 3-4 days. (3) Observe the swelling of the lateral buds under a microscope, cut off the swollen lateral buds, transfer them to CIM (MS+12mg / L chlorpyrifos) medium, and culture in the dark at 28°C for 16-18 days. (4) Distinguish the formed somatic embryos from non-somatic embryos, remove the non-somatic embryos, and transfer the somatic embryos to new CIM medium for further culture for 10-14 days. Note that fan-shaped embryos and torpedo embryos can be cultured for a maximum of 3 cycles. Too many cycles will affect the quality of the callus. (5) Divide the mature fan-shaped embryos and torpedo embryos into small clusters and transfer them to GD medium, with 8-12 clusters in each dish of GD medium. Culture them in the dark at 28°C for 21 days to induce the production of friable callus, i.e., FEC (Friable embryogenic callus). (6) Select friable callus and transfer them to new GD medium to maintain their vitality and use them in time to avoid prolonged culture time that may lead to a decrease in the vitality of friable callus. The remaining somatic embryos can be transferred to new GD medium for continued culture to induce the production of new friable callus.

[0123] 3. Transformation of cassava brittle callus

[0124] (1) Pipette 300 μL of recombinant Agrobacterium culture medium into 500 μL of fresh YEP liquid medium and activate the culture medium at 28°C, 200 rpm, until the culture medium becomes slightly turbid. (2) After activation, add 20 mL of YEP liquid medium for expansion. Culture conditions: 28°C, 200 rpm, culture until the OD of the culture medium is 600 Between 0.5 and 1.0. (3) Place the bacterial solution in a low-temperature centrifuge and centrifuge at 5000 rpm for 10 min at 4°C to collect the precipitate. (4) Wash the bacteria with GD liquid medium and centrifuge at 5000 rpm for 10 min at 4°C to collect the bacteria. Repeat once. (5) Resuspend the bacteria with GD liquid medium and take 1 mL of the resuspended bacterial solution to measure the OD 600. According to the formula A=0.20*20 / measured OD600 [A refers to the amount of resuspended bacterial solution absorbed, and GD (200μM AS) liquid culture medium is used to make up to 20mL], the OD600 value of the resuspended bacterial solution is diluted to 0.2. (6) Transfer the brittle callus to a sterile 50mL centrifuge tube, blow the callus apart, centrifuge at 1000rpm for 10min at room temperature, and then transfer to 28℃ and infect on a shaker at 40rpm for 25min. (7) Pour the callus onto absorbent paper covered with sterilized nylon mesh (100μM), spread it flat and dry it. Make sure the water is fully dried, otherwise it will affect subsequent growth. (8) Transfer the brittle callus and the nylon mesh to GD (200μM AS) solid culture medium and culture them in the dark at 22℃ for 2-3 days. (9) After co-cultivation, the callus was transferred to a sterile 50 mL Erlenmeyer flask and washed 2-3 times with GD (500 mg / L Carb) liquid medium. The washed callus was still poured onto absorbent paper covered with sterile nylon mesh (100 μM), spread flat and air-dried. (10) The brittle callus was transferred to GD (250 mg / L Carb) solid medium and cultured at 28°C with a 16-hour photoperiod for 7 days. (11) The callus tissue was transferred to GD medium (250 mg / L Carb + 5 mg / L Hyg) with more antibiotics and cultured at 28°C for one week with a 16-hour photoperiod. (12) The callus tissue was transferred to the screening medium (MSN + 250 mg / L Carb + 5 mg / L Hyg) and cultured at 28°C for one week with a 16-hour photoperiod. (13) Move the callus to a medium with a higher screening concentration (MSN+250mg / L Carb+8mg / L Hyg), and culture at a constant temperature of 28°C for one week with a photoperiod of 16 hours. (14) Then transfer the callus to a medium with a higher screening concentration (MSN+250mg / L Carb+15mg / L Hyg), and culture at a constant temperature of 28°C for one week with a photoperiod of 16 hours. (15) Repeat step (14) until cotyledons grow. (16) Pick the callus with cotyledons and transfer it to CEM (100mg / L Carb+0.1mg / L 6-BA) medium, and culture at 28°C with a photoperiod of 16 hours for 14 days. (17) Repeat step (16) until the cotyledons root. (18) Transfer the rooted cotyledons to MS medium, and culture at 28°C with a photoperiod of 16 hours until seedlings grow.

[0125] 4. DNA extraction and PCR detection of transgenic plants

[0126] The leaves of the transgenic cultured seedlings with good growth were collected and the leaf DNA was extracted by CTAB method. 2×CTAB extract was purchased from Beijing Coolable Technology Co., Ltd. The DNA of the transgenic cassava plant was used as a template, and the hygromycin universal detection primer on the vector and the universal primer on the vector formed a new pair of detection primers for PCR detection. If the target band appeared in the electrophoresis detection of the PCR product and the sequencing result was correctly compared, it was proved that the plant was a transgenic positive plant.

[0127] After the cotyledons were transferred to the MS medium for culture, the transgenic cassava plants were observed every week, and the leaf morphology, color, rooting status, plant height, etc. of the transgenic cassava plants were recorded with a camera. Figure 8 As shown, after 8 weeks of cotyledon cultivation, the leaves of non-transgenic cassava plants were emerald green, the plant height was 13 cm, and the root growth was lush; the leaves of overexpressing transgenic cassava plants were emerald green, but the plant height was only 3 cm, and the root growth was poor.

[0128] 10. Analysis of the function of cassava MeChlD gene based on VIGS technology

[0129] 1. Construction of VIGS vector pCsCMV-NC-MeChlD

[0130] The specific fragment shown in SEQ ID NO: 2 (the 300bp-554bp of the sequence shown in SEQ ID NO: 1) was selected from the cassava MeChlD gene sequence as the silencing fragment, and primers were designed, and 20bp of the universal linker sequence of the NC vector (indicated by lowercase letters) was added to both ends of the primers, as shown below:

[0131] MeChlD F:agtggtctctgtccagtcctAACTGCCTTGTTGCTTGG;

[0132] MeChlD R: ggtctcagcagaccacaagtAGACCCAATGAGTCTGTCC. Using the MeChlD gene as a template, primers MeChID CM-F and MeChID CM-R were used for PCR amplification, and the amplified product was recovered. The PCR reaction system was 2× Max Master Mix (Dye Plus) 25 μL, upstream primer (10 μM) 2 μL, downstream primer (10uM) 2 μL, template cDNA 2 μL, ddH2O to 50 μL. PCR amplification program: 95℃5min; 95℃30s, 55℃30s, 72℃40s, 35 cycles; 72℃7min. The sequence of the amplified product without the vector adapter is shown in SEQ ID NO: 3.

[0133] The recovered sequence fragments were connected to the NC system universal expression vector using the NC vector seamless cloning method. The connection system is shown in Table 5.

[0134] Table 5 NC cloning reaction system

[0135]

[0136]

[0137] First, mix the PCR recovery product with the pCsCMV-NC empty plasmid, add 5μL Mix, pipette 10-20 times, mix thoroughly, react at 50℃ for 30-60min, transform 2-5μL of the reaction product into E. coli competent DH 5α, then pick a single clone for overnight culture, bacterial liquid PCR identification, and positive clone sequencing. If the final sequencing result is correctly aligned, it means that the vector construction is complete. The constructed recombinant vector needs to be transformed into GV3101-pSoup-p19 Agrobacterium competent for storage.

[0138] 2. VIGS vector injection into plants

[0139] The Agrobacterium culture containing pCsCMV-NC-MeChlD recombinant plasmid and pCsCMV-NC empty plasmid were transferred to YEP medium containing 25 μg / mL rifampicin and 50 μg / mL kanamycin, respectively, and placed at 28°C and 200 rpm shaking on a shaker for activation. When the culture solution was slightly turbid, it could be transferred to a 50 mL Erlenmeyer flask for expansion and culture to OD 600 The bacterial cells were collected by low-speed centrifugation (5000 rpm) for 10 min, and then the bacterial liquid was resuspended in infection liquid (the formula is the same as that of tobacco infection liquid) to make the OD of the bacterial liquid 600 The value is 0.8-1.0. Then, a 1mL syringe was used to inoculate the leaves of the plants. Each plant was inoculated with 6-8 mature leaves, 4-6 sites were injected on each leaf, and 0.5-1mL of bacterial solution was injected on each leaf. Plants inoculated with Agrobacterium containing pNC-CsCMV empty vector were used as controls. After the injection, the plants were moved to a dark room for overnight culture, and then placed outdoors for normal culture. The growth and phenotypic changes of the plants were observed every 3-4 days. After obvious morphological changes appeared, the leaves were collected to extract DNA for PCR verification to obtain transgenic positive plants.

[0140] 3. VIGS plant phenotype detection

[0141] Cassava plants with good growth and undamaged leaves were selected for injection inoculation. After infection, the growth of the plants was observed every 3-4 days. It was found that the phenotype of cassava leaves changed significantly 3 weeks after inoculation. The leaf morphology of the inoculated plants was photographed with a camera. The results are as follows: Fig. 9As shown: 3 weeks after inoculation, the young leaves (sprouted after inoculation) of cassava plants inoculated with pCsCMV-NC-MeChlD silencing vector turned light green without purple-red, and the leaves became thinner; the young leaves of cassava plants inoculated with pCsCMV-NC empty vector were emerald green with obvious purple-red. 5 weeks after inoculation, the young leaves of the inoculated plants gradually matured, and the leaf colors were significantly different. The new leaves of cassava plants inoculated with pCsCMV-NC empty vector (developed from the young leaves after inoculation) were all emerald green, while the new leaves of cassava plants inoculated with pCsCMV-NC-MeChlD silencing vector showed large-scale chlorosis, and the leaves turned yellow partially or as a whole, indicating that the chlorophyll content in the leaves may be greatly reduced due to the reduced expression of the gene MeChlD.

[0142] 4. Determination of chlorophyll content in VIGS plants

[0143] Chlorophyll was extracted by acetone-ethanol extraction method. Three silent plants were randomly selected, and three mature leaves were taken from each plant. 0.2 g of the leaf midrib was taken, cut into pieces and put into a 25 mL brown volumetric flask, and 25 mL of extracting solution (acetone: ethanol = 1:1) was poured in, and the extract was placed in a 40°C incubator in the dark for 24 hours. The absorbance value of the filtrate was measured by ultraviolet spectrophotometer, and the main measured wavelengths were 645 nm and 663 nm. The following formula is the calculation formula for chlorophyll components:

[0144] Chlorophyll a (mg / g) = (12.7D663-2.69D645) * V / W

[0145] Chlorophyll b (mg / g) = (22.9D645-4.68D663) * V / W

[0146] Total chlorophyll (mg / g) = (20.21D645 + 8.02D663) * V / W

[0147] Where D663 and D645 are the absorbance values ​​of chlorophyll solution at wavelengths of 663nm and 645nm; V is the volume of the extract (L); W is the fresh weight of the material (g).

[0148] The results are as follows Fig.10 As shown: Compared with cassava plants injected with an empty vector (pCsCMV-NC), the contents of chlorophyll a and chlorophyll b in the new leaves of the silenced plants pCsCMV-NC-MeChlD were extremely significantly reduced (P>0.001); the ratio of chlorophyll a / b was extremely significantly increased (P>0.001); and the total amount of chlorophyll was extremely significantly reduced (P>0.001).

[0149] 5. VIGS plant silencing efficiency detection and gene expression identification

[0150] The silencing efficiency of the gene MeChlD in VIGS plants was analyzed by real-time quantitative PCR. After VIGS inoculation, 0.1-0.2 g of new leaves were collected, and the total RNA of the leaves was extracted using the RNAprep Pure Plant Plus Kit from Tiangen Company. The reverse transcription was performed using the TransScript One-Step gDNA Removal and cDNA Synthesis Super Mix kit purchased from Beijing Quanshijin Biotechnology Company. The expression of the ChlD gene was detected by real-time quantitative PCR. The primers for detecting the silencing efficiency of MeChlD are shown in Table 4. β-Actin was used as the internal reference gene, and the quantitative detection was performed using the Real-time Thermal Cycler fluorescence quantitative instrument of Thermo Fisher Company. The PCR reaction system was 2×ChamQUniversal SYBR qPCR Master Mix*5μL, upstream primer 0.4μL, downstream primer 0.4μL, template cDNA 1μL, and DNase-Free Water 3.2μL. The PCR amplification program was as follows: 95°C for 7 min; 95°C for 5 s, 60°C for 30 s, 60°C for 30 s, for a total of 40 cycles; 60°C for 1 min.

[0151] result( Fig.11 ) showed that compared with the empty control pCsCMV-NC, the relative expression level of the ChlD gene in cassava plants injected with the silencing vector pCsCMV-NC-MeChlD was 30%-60% of that of the empty control, that is, the silencing efficiency was 70%-40%, which was extremely significant (P<0.001), indicating that the silencing vector pCsCMV-NC-MeChlD can effectively silence the MeChlD gene in cassava.

[0152] The expression levels of other photosynthesis-related genes in cassava, including MeChlM, MeChlI, MeChlH, MePEPCase, MeRbcS, MeRbcL, MePrx-Q, etc., in plants with MeChlD gene silenced were detected by real-time quantitative PCR. The detection primers are shown in Table 4, and the detection method is the same as the silencing efficiency detection method. The results are shown in Fig.12 In the silenced plants, except for MeRbcL and MeChlH genes, which showed an increasing trend, the expression levels of other genes showed a decreasing trend, and the decreasing range was MePEPCase>MeRbcS>MePrx-Q>MeChlM>MeChlI.

[0153] 6. Field phenotypic identification of VIGS silenced plant MeChlD-2 and determination of root number, tuber fresh weight and total starch content of root tubers

[0154] result( Fig.13 ) showed that 4 months after transplanting to the field, the leaves of the VIGS-silenced plant MeChlD-2 still retained mosaic or yellowing. At the same time, after the cassava tubers were harvested, it was found that the number of tubers, fresh tuber weight and total starch content in the VIGS-silenced plant MeChlD-2 were significantly lower than those in the control plant. This shows that MeChlD is not only involved in photosynthesis (source), but also affects the accumulation of starch in cassava tubers (sink).

[0155] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modification and substitution of the practical is also within the scope of the present invention. Therefore, the equalization transformation and modification made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. Cassava MeChlD Gene, or containing cassava MeChlD Application of a recombinant vector of a coding region sequence of a gene in improving the ability of a prokaryotic expression strain to resist magnesium stress, the cassava MeChlD The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. Overexpression of Cassava MeChlD Gene, or containing the cassava MeChlD Application of gene overexpression vector or host bacteria in regulating cassava plant height and root growth, overexpressing the cassava MeChlD The gene, or the vector or host bacteria transforming cassava can make the cassava plant dwarfed and the root growth poor. MeChlD The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

3. Use of a silencing gene fragment, or a recombinant vector or host bacteria containing the silencing gene fragment in reducing the number of cassava tubers, and / or fresh tuber weight, and / or total starch content of cassava tubers, wherein the nucleotide sequence of the silencing gene fragment is shown in SEQ ID NO:

2.

4. The silencing gene fragment, or the recombinant vector or host bacteria containing the silencing gene fragment improves LqCy Gene expression and / or enhancement MeChl Gene expression and / or reduction MeChlD Gene expression and / or reduction MeChlI Gene expression and / or reduction MeChl Gene expression and / or reduction MePEPCase Gene expression and / or reduction ikB Gene expression and / or reduction QUR In the application of gene expression, the nucleotide sequence of the silencing gene fragment is shown as SEQ ID NO: 2.

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