CN118064403A - Phyllostachys Pubescens pectin acetyl esterase gene PePAE and extraction method and application thereof - Google Patents
Phyllostachys Pubescens pectin acetyl esterase gene PePAE and extraction method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a phyllostachys pubescens pectin acetyl esterase gene PePAE and an extraction method and application thereof. The nucleotide sequence of the gene is shown as SEQ ID NO. 1. The gene has the functions of reducing the acetylation level of pectin on plant cell walls, promoting pectin accumulation on plants, improving photosynthetic capacity of the plants and promoting plant growth; the phyllostachys pubescens pectin acetylesterase gene PePAE is introduced into rice for verification, so that the transgenic rice plant is lower in cell wall pectin acetylization level, higher in pectin accumulation amount, stronger in leaf photosynthetic capacity and faster and better in plant growth than the wild rice plant.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a phyllostachys pubescens pectin acetyl esterase gene PePAE and an extraction method and application thereof.
Background
Pectin is a class of galacturonic acid-rich branched heteropolysaccharides that are mainly found in the primary and middle layers of type i plants, and in small amounts in the primary and secondary walls of type ii plants. Pectin consists of four major classes of components, namely polygalacturonic acid (homogalacturonan, HG), rhamnogalacturonan I (rhamnogalacturonan I, RGI), rhamnogalacturonan ii (rhamnogalacturonan ii, RG ii) and xyloglucan galacturonic acid (xylogalacturonan, XGA); the four main components mainly participate in the composition matrix, so that the primary wall has elasticity and can be prolonged along with the elongation of cells, and also participate in the remodelling of cell walls and the expansion of cells, thereby further affecting or changing the plant ontogeny phenotype and physiological and biochemical processes.
The pectin content is very large in the primary wall of dicotyledonous plants, accounting for about one third of the dry weight; the herbs in monocotyledonous plants are then relatively low, for example grasses. However, there are still a large number of genes involved in pectin synthesis and modification in monocot genomes, which suggests that pectin also plays an important role in the individual growth and development of monocots; non-sugar groups are common groups that modify pectin, such as methyl (METHYLESTERS, me) and acetyl (ACETYLESTERS, ae) esters, etc., which allow pectin to be synthesized in a highly esterified state; and various modification enzymes obtained by modification of the modification groups regulate the esterification modification degree of plant components, change physicochemical properties such as plant viscosity, hydrophobicity, charging characteristics and the like, regulate structural properties such as hardness, ductility, porosity and the like of the whole plant cell wall and participate in the dynamic regulation activity of the plant cell wall, and respond various stimulation and release signal substances so as to adapt to different growth and development stages of different tissues of the plant cell wall. Among them, acetyl modification of pectin is a common modification of polysaccharide components in plant cell walls and is also an important modification of pectin.
As plant pectin which can cleave acetyl ester bonds to deacetylate pectin, acetyl esterase (PECTIN ACETYLESTERASE, PAE; E.C.3.1.1.6) largely determines the level of acetylation of pectin and cell walls. According to CAZy database, PAE belongs to the CE13 family of carbohydrate esterases (Carbohydrate Esterases, CEs). Following isolation of the first plant PAE from Citrus sinensis, related studies have been widely developed in arabidopsis thaliana (ArabidoPsis thaliana), poplar (PoPulustrichocarPa), tea tree (CAMELLIA SINENSIS) and the like. For example, the division of the AtPAE members of the 12 PAE genes into 3 branches in arabidopsis, 3 of the 9 conserved motifs (GCSxG, nxayDxwQ and HCQ) of AtPAEs are presumed to be possibly associated with a catalytic mechanism, and the expression pattern of AtPAEs was found to be consistent with the genes related to pectin methylesterase, pectin lyase, polygalacturonase and the like, but the specific function was unknown; studies on report mutants Pae8 and Pae9 in Arabidopsis indicate that the PAE8 and PAE9 proteins simultaneously play a role in removing one-third of cell wall acetyl esters in the pectin formation process of Arabidopsis leaves, so that the accumulation amount of the cell wall acetyl esters is greatly reduced, and the reduction of the flower stem height is further shown; meanwhile, the research on PAE (pallet-like) such as poplar, tea tree, sweet orange and the like shows that the plant PAE has high conservation among different species, and only has an independent and typical PAE conservation domain; furthermore, PAE expression was found to be regulated in poplar by CO 2 and nitrogen concentration levels; the CsPAE in the sweet orange can improve the resistance of plants to bacillosis, which indicates that the plant PAE has functional diversity.
As one of grass plants of the family poaceae with high economic value, bamboos have edible shoots and highly lignified bamboo stalks. As the moso bamboo in the class of bamboos, which is a representative of fast-growing plants, the rapid growth of moso bamboo is closely related to the remodeling of cell walls and the dynamic optimization of cell wall polysaccharide properties. Pectin, as a cell wall network structure matrix material, is an indispensable component in the cell expansion and elongation process, and is one of the basic conditions for rapid growth of plants. At present, no related prior art exists on the mechanism of acetylation and deacetylation of bamboo cell wall pectin, and the influence of PAE on the whole cell wall and the physiological and biochemical activities of plants caused by the PAE is also blank. Therefore, the identification of PAE in bamboo and the disclosure of its molecular characteristics are of great importance in elucidating the biological functions of PAE in bamboo growth and development.
Disclosure of Invention
Aiming at the problems, the pectin acetyl esterase gene PePAE is extracted from phyllostachys pubescens, and the gene has the functions of reducing the acetylation level of pectin on the cell wall of a plant, promoting pectin accumulation of the plant, improving the photosynthetic capacity of the plant and promoting the growth of the plant, and can be used as a target gene to cultivate the plant with low acetylation level of pectin on the cell wall, high pectin content, strong photosynthetic capacity and fast growth through a transgenic means.
In order to achieve the above purpose, the present invention may adopt the following technical scheme:
the invention provides a phyllostachys pubescens (Phyllostachys edulis) pectin acetyl esterase gene PePAE, and the nucleotide sequence of the phyllostachys pubescens (Phyllostachys edulis) pectin acetyl esterase gene is shown as SEQ ID NO. 1.
In another aspect, the invention provides an isolated nucleic acid molecule encoding a nucleotide sequence of the amino acid sequence shown as SEQ ID NO. 2.
In a further aspect the invention provides a biological material comprising the phyllostachys pubescens pectin acetylesterase gene PePAE of the invention or an isolated nucleic acid molecule of the invention.
In a further aspect, the invention provides the use of the phyllostachys pubescens pectin acetylesterase gene PePAE of the invention or the isolated nucleic acid molecule of the invention or the biomaterial of the invention in one or more of the following applications: (a) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or an isolated nucleic acid molecule or biological material in reducing plant cell wall pectin acetylation level; (b) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or an isolated nucleic acid molecule or biological material in promoting pectin accumulation in plants; (c) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or an isolated nucleic acid molecule or biological material in promoting photosynthesis of plants; (d) The application of phyllostachys pubescens pectin acetylesterase gene PePAE or an isolated nucleic acid molecule or biological material in promoting plant growth.
In a further aspect, the invention provides the use of the phyllostachys pubescens pectin acetylesterase gene PePAE of the invention or the isolated nucleic acid molecule of the invention or the biomaterial of the invention for the cultivation of transgenic plants.
In still another aspect, the present invention provides a method for extracting phyllostachys pubescens pectin acetylesterase gene PePAE in the present invention, which comprises: (1) Extracting RNA from phyllostachys pubescens leaves, and reversely transcribing the RNA into cDNA as a template; (2) And (3) performing PCR amplification by using a primer pair to obtain the phyllostachys pubescens pectin acetylesterase gene PePAE, wherein the upstream primer of the primer pair is shown as SEQ ID NO. 3, and the downstream primer of the primer pair is shown as SEQ ID NO. 4.
The beneficial effects of the invention at least comprise: the pectin acetyl esterase gene PePAE of the phyllostachys pubescens has the functions of reducing the pectin acetylation level of plant cell walls, promoting pectin accumulation of plants, improving the photosynthetic capacity of the plants and promoting the growth of the plants; the phyllostachys pubescens pectin acetylesterase gene PePAE is introduced into rice for verification, so that the transgenic rice plant is lower in cell wall pectin acetylization level, higher in pectin accumulation amount, stronger in leaf photosynthetic capacity and faster and better in plant growth than the wild rice plant.
Drawings
FIG. 1 is a diagram of a double-enzyme-cut agarose gel electrophoresis of the recombinant plasmid pCAMBIA1300-Ubi-PePAE of example 2, wherein M1: DNA molecular weight marker DL15000;1-3: an expression plasmid;
FIG. 2 is a map of the pCAMBIA1300-Ubi-PePAE expression vector of example 2;
FIG. 3 is a PCR electrophoretogram of a monoclonal colony of the expression vector pCAMBIA1300-Ubi-PePAE plasmid-transformed Agrobacterium of example 3, wherein M2: DNA molecular weight marker DL5000;1: positive control with pCAMBIA1300-Ubi-PePAE plasmid as template; 2: a negative control with pCAMBIA1300-Ubi empty vector plasmid as a template; 3: negative control with water as template; 4-8: monoclonal colonies transformed with pCAMBIA 1300-Ubi-PePAE;
FIG. 4 is an electrophoresis chart of PCR detection results of PePAE in rice plants of example 4, wherein M3: DNA molecular weight marker DL5000;1-3: over-expressing PePAE DNA extracted from rice line OE-1; 4-6: over-expressing PePAE DNA extracted from rice line OE-2; 7-9: DNA extracted from wild rice; 10: negative control with water as template; 11: a negative control with pCAMBIA1300-Ubi empty vector plasmid as a template; 12: positive control with pCAMBIA1300-Ubi-PePAE plasmid as template;
FIG. 5 is a representative picture of wild type rice and over-expressed PePAE rice lines OE-1 and OE-2 in example 4;
FIG. 6 is a bar graph of comparative analysis of the acetylation level of PePAE transgenic rice cell wall pectin in example 5, showing significant differences at levels 0.01< P.ltoreq.0.05, in terms of amount of acetic acid released after saponification in the extract;
FIG. 7 is a comparative analysis bar graph of pectin content in PePAE transgenic rice in example 6, showing significant differences at a level of 0.01< P.ltoreq.0.05, based on the content of galactosylate as the main pectin component in the extract;
FIG. 8 is a bar graph of net photosynthetic rate analysis of PePAE transgenic rice, example 7, showing significant differences at a level of 0.01< P.ltoreq.0.05;
FIG. 9 is a bar graph of the concentration analysis of intracellular carbon dioxide in PePAE transgenic rice in example 7, showing significant differences at 0.01< P.ltoreq.0.05 and showing significant differences at P.ltoreq.0.01;
FIG. 10 is a bar graph of the air pore conductance analysis of the transgenic rice plant of dry PePAE6 in example 7, showing significant differences at a level of 0.01< P.ltoreq.0.05;
FIG. 11 is a bar chart showing the analysis of the increase in seedling height (overground growth) over 2 weeks for PePAE transgenic rice in example 8, showing significant differences at P.ltoreq.0.01 level;
FIG. 12 is a bar chart showing analysis of root length increment (amount of growth of the subsurface portion) of PePAE6 transgenic rice in example 8 over 2 weeks, n.s. represents P >0.05;
FIG. 13 is a bar graph of the fresh weight analysis of the aerial parts of PePAE transgenic rice plants in example 8, showing significant differences at P.ltoreq.0.01 level;
FIG. 14 is a bar graph of upper dry weight analysis of PePAE transgenic rice plants of example 8 showing significant differences at P.ltoreq.0.01 levels;
FIG. 15 is a bar graph of the fresh weight analysis of the subsurface of PePAE transgenic rice plants in example 8, showing significant differences at P.ltoreq.0.01 level;
FIG. 16 is a bar graph of the lower dry weight analysis of PePAE transgenic rice plants of example 8 showing significant differences at P.ltoreq.0.01 levels.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context clearly differs, singular forms of expression include plural forms of expression. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to indicate the presence of a feature, number, operation, component, part, element, material, or combination. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, elements, materials or combinations thereof may be present or added. As used herein, "/" may be interpreted as "and" or "as appropriate.
The embodiment of the invention provides a phyllostachys pubescens (Phyllostachys edulis) pectin acetyl esterase gene PePAE, and the nucleotide sequence of the phyllostachys pubescens (Phyllostachys edulis) pectin acetyl esterase gene is shown as SEQ ID NO. 1.
Another embodiment of the invention provides an isolated nucleic acid molecule encoding a nucleotide sequence of the amino acid sequence shown as SEQ ID NO. 2.
It should be noted that the nucleotide sequences capable of encoding the amino acid sequence shown in SEQ ID NO. 2 are all sequences protected by the present application. For example, the sequence shown in SEQ ID NO. 1 can be optimized. Specifically, the optimization aims at improving the expression efficiency of the nucleotide sequence, and the optimization method is a technical means existing in the field and mainly comprises the following means: (1) Such as the design and modification of expression vectors for the transcription stage, e.g., the use of highly efficient expression elements such as promoters, enhancers, etc.; (2) The improvement of translation stage, the optimization of gene coding sequence of recombinant protein itself, including codon bias optimization; (3) Optimizing host cells, including efficient expression site positioning and targeted recombination; and (4) optimizing the large-scale cell culture process. The sequence shown in SEQ ID NO. 1 can be optimized by a person skilled in the art according to specific needs. In some embodiments, the promoter or enhancer may be Ubipromoter or CaMV35Spromoter (enhanced); for codon optimisation, briefly, due to the degeneracy of the codons, and the preference of different species for different codons encoding the same amino acid during translation, and the influence on mRNA stability, translation efficiency, etc., the "optimal codons", "codon optimisation" have been developed, and specific optimisation methods can be seen "Presnyak,Vladimir et al."Codon optimality is a major determinant ofmRNA stability."Cell.2015Mar 12;160(6):1111-1124.".
Specifically, the sequence of SEQ ID NO:1 is shown below:
ATGAAGACGAGGGAGAATGTTGGGATCACCAAATCCTGGGCTCATCTTCTTCTCCTTCTTGTGGTTGTTCTTGTCCTTGTGAGAAGCAGCGCGCAGGCGGCGGCCGACGAGCACAAGATCGGCGGCAGTAGGAGGCGGCGTGCGGCGGCGGCGGCGGCGCCGCCCGTTATGGTGCCTATCACCCTCCTCAGATCAGCCGTCGACAAGGGAGCTGTGTGCATGGATGGGACGCCGCCTGCTTACCACTTGGACCATGGCTCCGGGGCAGGGAACAGCAGCTGGATGATATTCCTAGAGGGAGGCGGGTGGTGCAACGACGTGTGGTCGTGCCGGTACCGTGCGGCGAGCCGGCTGGGCTCGTCGGATCGCATGGAGAAGCAGATCTACTTCGGGGGCATCAGGAGCGCCAACCCCCTCGACAACCCCGATTTCTACAACTGGAACCGGGTGATGATTCGCTACTGCGACGGCGCGTCCTTCGCCGGCGAAGGCTTCGACAAGGATCATGGGTTCTATTTCCGGGGCCAGCGCATCTGGGACGCGGTCGTCCGGCACCTCCTCTCCATCGGAATGGCCTCTGCAGATCAGGTGTTGCTCACCGGCGCCTCCGCCGGTGGACTGGCGGCCATCCTGCACTGCGACCAGTTCAGAGCCTTCTTCCCCGCCGCCACTGCCGGCGGCCGGAGCACCACCGTCAAGTGCCTCGCCGACGCAGGCCTCTTCCTCGACGCCGTGGATGTCTCCGGGGGCCGCAGCTTGAGATCGTACTACGGAGACGTCGTAGCCATGCAGGGGGTAGCTCAGAACCTGCCGCCGACTT GCACCGACCATCTGGACGCCACCTCGTGCTTCTTCCCTCAGAATATAATCGATGGCATAAACACCCCAATCTTCCTGCTAAATGCAGCATACGATGTCTGGCAGATCCAGCAAAGTTTGGCCCCAAACAAAGCTGACCCCAGCGGCGCCTGGCGAGCCTGCAAGTTCAACCGCTCAGCCTGCAATGCATCCCAGATGAAGTTCTTTCAAGAATTCAGGGGCCAGATGATAGCATCTGTGAAAGGTTTCTCCAGTTCCAAGAGCAACGGGTTGTTCATAAACTCGTGCTTCACTCACGGCCAGTCTGAGGCACCGGCCACCTGGAATAGTGCAGCTGGCTCTCCTGCTATTCAAAACAAGGGGATTGCAAAATCTGTTGGTGACTGGTACTTTGGTCGGGCTGAAGTGAAGGCGATCGACTGCCCTTATCCCTGCGACAAAACATGCCGTCACGACATA;
SEQ ID NO. 2 sequence is shown below:
MKTRENVGITKSWAHLLLLLVVVLVLVRSSAQAAADEHKIGGSRRRRAAAAAAPPVMVPITLLRSAVDKGAVCMDGTPPAYHLDHGSGAGNSSWMIFLEGGGWCNDVWSCRYRAASRLGSSDRMEKQIYFGGIRSANPLDNPDFYNWNRVMIRYCDGASFAGEGFDKDHGFYFRGQRIWDAVVRHLLSIGMASADQVLLTGASAGGLAAILHCDQFRAFFPAATAGGRSTTVKCLADAGLFLDAVDVSGGRSLRSYYGDVVAMQGVAQNLPPTCTDHLDATSCFFPQNIIDGINTPIFLLNAAYDVWQIQQSLAPNKADPSGAWRACKFNRSACNASQMKFFQEFRGQMIASVKGFSSSKSNGLFINSCFTHGQSEAPATWNSAAGSPAIQNKGIAKSVGDWYFGRAEVKAIDCPYPCDKTCRHDI.
It is noted that in some embodiments, the amino acid sequence encoded as shown in SEQ ID NO.2 includes a stop codon, such as "TGA", at the end of the sequence shown in SEQ ID NO. 1.
Yet another embodiment of the present invention provides a biological material which may comprise the phyllostachys pubescens pectin acetylesterase gene PePAE of the invention or an isolated nucleic acid molecule of the invention.
It should be noted that the phyllostachys pubescens pectin acetyl esterase gene PePAE in the present invention or the separable nucleic acid molecule in the present invention can be prepared into a product more favorable for practical application. For example, the gene expression cassette, the gene expression vector, the gene cloning vector, engineering bacteria or engineering cells can be prepared; the forms of gene expression cassettes, gene expression vectors, gene cloning vectors, engineering bacteria or engineering cells are known in the art. For example, the engineering bacteria can be agrobacterium commonly used in plant cell genetic engineering, and the phyllostachys pubescens pectin acetyl esterase gene PePAE of the invention or the separable nucleic acid molecule of the invention is transduced into agrobacterium to prepare engineering bacteria for expressing the gene PePAE or the nucleic acid molecule; for another example, the engineered cell is typically a tissue cell or fertilized egg of a plant.
Yet another embodiment of the invention provides a use of the phyllostachys pubescens pectin acetylesterase gene PePAE of the invention or an isolated nucleic acid molecule of the invention or a biological material of the invention in one or more of the following applications: (a) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or an isolated nucleic acid molecule or biological material in reducing plant cell wall pectin acetylation level; (b) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or an isolated nucleic acid molecule or biological material in promoting pectin accumulation in plants; (c) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or an isolated nucleic acid molecule or biological material in promoting photosynthesis of plants; (d) The application of phyllostachys pubescens pectin acetylesterase gene PePAE or an isolated nucleic acid molecule or biological material in promoting plant growth.
In some embodiments, the application (3) may include one or more of the following applications: (a) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or an isolated nucleic acid molecule or biological material in improving net photosynthetic rate of plants; (b) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or an isolated nucleic acid molecule or biological material in improving the concentration of carbon dioxide between plant cells; (c) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or isolated nucleic acid molecules or biological materials in improving plant stomata conductivity; or the application (4) may comprise one or more of the following applications: (a) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or isolated nucleic acid molecules or biological materials in increasing plant overground growth; (b) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or isolated nucleic acid molecules or biological materials in increasing plant underground growth; (c) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or an isolated nucleic acid molecule or biological material in increasing fresh weight of plants; (d) Use of phyllostachys pubescens pectin acetylesterase gene PePAE or an isolated nucleic acid molecule or biological material to increase plant dry weight.
It should be noted that, as proved by using rice in the invention, the pectin content in the transgenic rice leaves transformed by using the phyllostachys pubescens pectin acetylesterase gene PePAE is 119.76% and 120.25% of the wild rice respectively. The pectin accumulation level of the over-expressed PePAE strain OE-1 and OE-2 plants is improved compared with that of the wild rice. It should be noted that, the index of decreasing the acetylation level of the pectin on the plant cell wall includes, in particular, increasing the accumulation amount of plant acetic acid, that is, pePAE, the product produced by the catalytic reaction during decreasing the acetylation level of the pectin on the plant cell wall; the leaf extracts of transgenic rice transformed with the phyllostachys pubescens pectin acetylesterase gene PePAE were saponified to release acetic acid in 86.87% and 87.52% of WT, respectively. The cell wall pectin acetylation level of the over-expressed PePAE strain OE-1 and OE-2 strain is lower than that of the WT; the net photosynthetic rate of the transgenic rice transformed by using the phyllostachys pubescens pectin acetylesterase gene PePAE is 115.92% and 118.05% of the wild type rice, the concentration of intercellular carbon dioxide is 102.84% and 104.02% of the wild type rice, and the air pore conductivity is 138.31% and 143.04% of the wild type rice. The photosynthesis ability of the transgenic rice transformed by using the phyllostachys pubescens pectin acetylesterase gene PePAE is promoted compared with that of a wild rice type; the upper-ground growth amounts of the transgenic rice transformed by using the phyllostachys pubescens pectin acetylesterase gene PePAE are 114.08% and 111.03% of the wild rice, the lower-ground growth amounts of the transgenic rice are 109.62% and 112.82% of the wild rice, the biomass of the transgenic rice transformed by using the phyllostachys pubescens pectin acetylesterase gene PePAE6 is increased, the fresh weight of the upper-ground parts is 232.28% and 274.05% of the wild rice, the dry weight of the upper-ground parts is 212.63% and 251.60% of the wild rice, the fresh weight of the lower-ground parts is 262.28% and 300.33% of the wild rice, and the dry weight of the lower-ground parts is 131.59% and 110.66% of the wild rice, respectively, so that the phyllostachys pubescens pectin acetylesterase gene PePAE can promote plant growth. In particular, aspects of promoting plant growth include increasing plant growth and increasing plant biomass.
It should be further noted that, based on the fact that the phyllostachys pubescens pectin acetylesterase gene PePAE has the functions of reducing the pectin acetylation level of the plant cell wall, increasing the pectin accumulation, enhancing the photosynthesis ability of leaves and promoting the growth of plants, the optimized separable nucleic acid molecules and the biological material loaded with the gene PePAE and the separable nucleic acid molecules have the functions as described above.
In a further embodiment, the invention provides the use of the phyllostachys pubescens pectin acetylesterase gene PePAE of the invention or the isolated nucleic acid molecule of the invention or the biological material of the invention for the cultivation of transgenic plants.
As described above, the phyllostachys pubescens pectin acetylesterase gene PePAE of the invention or the isolated nucleic acid molecule of the invention or the biological material of the invention has the functions of reducing the pectin acetylation level of plant cell walls, increasing the accumulation of pectin, enhancing the photosynthetic capacity of leaves and promoting plant growth; the phyllostachys pubescens pectin acetylesterase gene PePAE, nucleic acid molecules or biological materials can be transferred into plants for cultivation through transgenic technology (including gene editing technology), so that low-cell-wall pectin acetylation level plants, high-pectin biomass plants, high-photosynthetic capacity plants and high-growth plants can be obtained.
Specifically, the terms "low cell wall pectin acetylation level plant", "high pectin biomass plant", "high photosynthetic capacity plant" and "high growth plant" refer to transgenic plants obtained by using the phyllostachys pubescens pectin acetylesterase gene PePAE as a target gene for transgenesis, and under the same detection method, the detected corresponding indexes (including galacturonic acid content, acetic acid release amount, net photosynthetic rate, intercellular carbon dioxide concentration, stomatal conductivity, seedling height increment, root length increment, fresh and dry weight of overground parts, fresh and dry weight of underground parts) are superior to those of corresponding optimized transgenic plants compared with wild type plants under the same condition.
In some embodiments, the plant used in the above application may be phyllostachys pubescens or rice. It should be noted that the plants may be plants known in the art, such as moso bamboo or rice.
It should be understood that after optimizing the phyllostachys pubescens pectin acetylesterase gene PePAE to increase the expression efficiency according to the above method, the transcription level of the PePAE gene is artificially increased or decreased by constructing a vector and transferring the vector into the phyllostachys pubescens origin, and then the phyllostachys pubescens obtained by culturing can be transgenic phyllostachys pubescens.
In another embodiment of the present invention, a method for extracting phyllostachys pubescens pectin acetylesterase gene PePAE in the present invention may include: (1) Extracting RNA from phyllostachys pubescens leaves, and reversely transcribing the RNA into cDNA as a template; (2) And (3) performing PCR amplification by using a primer pair to obtain the phyllostachys pubescens pectin acetylesterase gene PePAE, wherein the upstream primer of the primer pair is shown as SEQ ID NO.3, and the downstream primer of the primer pair is shown as SEQ ID NO. 4.
It should be noted that, the extraction method of phyllostachys pubescens pectin acetyl esterase gene PePAE in the present invention may be performed by methods known in the art, such as PCR amplification extraction, and the primer extracted by PCR amplification may be designed according to a conventional design method in the art, preferably, the upstream primer is shown in SEQ ID No. 3, the downstream primer is shown in SEQ ID No. 4, and a large amount of phyllostachys pubescens pectin acetyl esterase gene PePAE may be obtained under amplification of the primer pair.
For a better understanding of the present invention, the content of the present invention is further elucidated below in connection with the specific examples, but the content of the present invention is not limited to the examples below.
Example 1 acquisition of coding region sequence of Phyllostachys Pubescens pectin acetylesterase Gene PePAE6
Extracting RNA from bamboo shoots of phyllostachys pubescens (Phyllostachys edulis) and reversely transcribing the RNA into cDNA as a template; the method specifically comprises the following steps: the obtained RNA was reverse transcribed into cDNA using a kit (Jian Dan organism, TR205-D, beijing) first, followed by a step of referring to PRIMESCRIPT RT reverse transcription kit (Takara, RR037A, japan);
Designing a specific primer by using a predicted Gene PH02Gene08079 in moso bamboo, and amplifying a coding region sequence by PCR, wherein the primer sequence is shown in the following table 1; amplification system and amplification procedure are referred to the instructions of 2X TAQ MASTER Mix (Dye Plus) (Northenzan, P112-01, nanjing).
TABLE 1PCR amplification primers
| Upstream primer | 5′-GCTGATATGAAGACGAGGGAGAATG-3′(SEQ ID NO:3) |
| Downstream primer | 5′-CTTCTCATCATATGTCGTGACGGC-3′(SEQ ID NO:4) |
And (3) detecting the PCR amplification product by agarose gel electrophoresis, cutting off a target band, purifying and recovering the target band, connecting the recovered DNA fragment to a pGEM-TEasy vector, converting escherichia coli DH5 alpha competent cells, screening by blue white spots, extracting positive cloning plasmids, performing enzyme digestion map analysis, and sequencing the monoclonal, wherein the insert fragment is 1281bp, as shown in SEQ ID NO. 1. Subsequently, several tens of thousands of genes in the genome are predicted in moso bamboo by using Blast on-line software for comparison, and the sequence similarity between SEQ ID NO. 1 and PH02Gene08079 is found to be the highest and is 95.44%.
Further through on-line comparison analysis of BlastP software, the highest (83.23%) consistency of the amino acid sequence (SEQ ID NO: 2) encoded by the gene with the PAE (XM_ 039958098.1) of switchgrass (Panicum virgatum), the consistency with the PAE (XM_ 025961332.1) of millet (Panicum hallii) is 82.42%, and the consistency with the PAE (XM_ 045111292.1) of highland barley (Hordeum vulgare) is 81.85%; analysis of the protein domain showed that the protein had a PAE enzyme-related conserved domain PAE (pfam 03083). As a result, the cloned gene encodes a pectin acetylesterase, which was designated PePAE6, and pGEM-T Easy vector containing the gene was pT-PePAE.
EXAMPLE 2 construction of plant expression vector carrying Phyllostachys Pubescens pectin acetylesterase Gene PePAE6
Designing an amplification primer with a homology arm and an enzyme cleavage site BamHI/Pml I according to the sequence shown in SEQ ID NO. 1, taking the plasmid with correct sequencing obtained in example 1 as a template after dilution by 40 times, using PrimeSTARMix high-fidelity enzyme amplification enzyme, and amplifying a fragment containing the enzyme cleavage site (BamHI/Pml I) and deoxyribonucleotide sequence of a phyllostachys pubescens PePAE coding region required by the construction of an expression vector by PCR; primer sequences are shown in table 2 below; amplification system and amplification program referenceMax DNA Polymerase (Takara, R045).
TABLE 2 primer sequences
The PCR amplified products were subjected to agarose gel electrophoresis, the target band was excised, purified and recovered, and the target gene and pCAMBI1300-Ubi vector enzyme fragment were ligated by using one-step cloning technique, and the reaction system (20. Mu.L) was as shown in Table 3 below.
Table 3 objective gene and pCAMBI1300-Ubi vector enzyme section cloning reaction system
| Component (A) | Volume of |
| 5 Xreaction buffer | 4.0μL |
| NovoRecPlus recombinant enzymes | 1.0μL |
| PCAMBI1300-Ubi enzyme cutting carrier | 1.0μL |
| Target fragment | 1.0μL |
| ddH2O | 13.0μL |
Adding all the reagents into a PCR tube according to the sequence, mixing uniformly, centrifuging, placing in a water bath kettle at 50 ℃ for 10min, converting escherichia coli DH5 alpha, extracting plasmids, and performing enzyme digestion map identification and sequencing verification, wherein the enzyme digestion map identification is shown in figure 1; the obtained recombinant expression vector was designated as pCAMBIA1300-Ubi-PePAE (see FIG. 2).
EXAMPLE 3 identification of monoclonal colonies containing the expression vector pCAMBIA1300-Ubi-PePAE6
The expression vector pCAMBIA1300-Ubi-PePAE constructed in example 2 was transferred into competent cells of Agrobacterium (Agrobacterium tumefaciens) strain GV3101 by freeze thawing, and single colonies grown on plates with kanamycin resistance (50 mg. Multidot.L -1) were picked for PCR identification. The PCR detection was performed using the primers (shown in Table 2) of example 2 with the monoclonal colony formed after the PePAE gene recombinant expression vector transformation as a template, and the recombinant plasmid pCAMBIA1300-Ubi-PePAE6 as a positive control, and the pCAMBIA1300-Ubi empty vector and water as negative controls.
The result is shown in figure 3, and the result of PCR electrophoresis of the agrobacterium monoclonal colony transformed by the expression vector pCAMBIA1300-Ubi-PePAE plasmid shows that the monoclonal colony contains a target gene fragment, and the agrobacterium monoclonal bacterial liquid obtained by shaking can be used for infection transformation experiments.
Example 4PePAE6 transformation of Rice and PCR detection
Using the bacterial liquid obtained in example 3, wild type rice (WT) (Oryza sativa L.spp. Japonica) calli were infected, and 2 transgenic rice lines were finally obtained by selection for continuous resistance (hygromycin 50 mg. L -1) and gene expression detection was performed.
DNA of the transgenic rice plant and the wild rice plant was extracted, respectively, and PCR detection was performed using the primers of example 2 by PCR method. The results are shown in FIG. 4, which shows that the target gene was detected in the transgenic rice plants, but not in the wild rice plants, demonstrating PePAE that the rice plants had been transformed with PePAE. After 3 weeks of cultivation (hydroponic) under suitable conditions (28 ℃ ±2 ℃ in an illumination incubator, 12 hours of illumination, 12 hours of dark illumination, and 60% relative humidity) in a wood village B rice nutrient solution (cool rice, NS1050, beijing)), seedlings of rice were obtained, which could be subjected to subsequent experiments, see fig. 5.
EXAMPLE 5PePAE analysis of cell wall pectin acetylation level in transgenic Rice
The results of measuring the acetate content of the product produced after saponification of the pectin extract from rice leaves in example 4 are shown in FIG. 6, and show that the product content in WT was 5.208g/100g, and that the product content in over-expressed PePAE lines OE-1 and OE-2 was 86.87% (4.524 g/100 g) and 87.52% (4.558 g/100 g) of WT, respectively. It was demonstrated that over-expression PePAE6 reduced the cell wall pectin acetylation levels of OE-1 and OE-2 plants.
EXAMPLE 6PePAE analysis of pectin content in transgenic Rice
The results of measurement of pectin content in rice leaves in example 4 are shown in FIG. 7, and show that galacturonic acid content in WT is 3.311. Mu. Mol/g, and that in over-expressed PePAE strain OE-1 and OE-2 is 119.76% (3.965. Mu. Mol/g) and 120.25% (3.982. Mu. Mol/g) of WT, respectively. It was demonstrated that over-expression PePAE6 increased pectin accumulation in OE-1 and OE-2 plants.
Example 7PePAE6 comparative analysis of the photosynthetic physiological parameters of transgenic Rice
As shown in FIG. 8, FIG. 9 and FIG. 10, the statistical analysis after detection of photosynthetic parameters of 3-week old rice seedlings revealed that the net photosynthetic rate in WT was 14.663. Mu. Mol m -2s- 1, the intercellular carbon dioxide concentration was 278.278. Mu. Mol - 1, the stomatal conductance was 0.234mol m -2s- 1, the net photosynthetic rates in over-expressed PePAE6 strains OE-1 and OE-2 were 115.92% (16.998. Mu. Mol m -2s- 1) and 118.05% (17.310. Mu. Mol m -2s-) of WT, respectively, the intercellular carbon dioxide concentrations were 102.84% (286.171. Mu. Mol - 1) and 104.02% (289.468. Mu. Mol - 1) of WT, and the stomatal conductance was 138.31% (0.324. Mu. Mol m -2s- 1) and 143.04% (0.335 mol m -2s- 1) of WT, respectively. It was demonstrated that over-expression PePAE6 increased photosynthetic capacity in OE-1 and OE-2 plants.
Example 8PePAE6 transgenic Rice phenotype and biomass comparative analysis
Statistical analysis was performed on physiological phenotypes of 3-week old rice seedlings, including seedling height increment (amount of overground part growth) over 2 weeks, root length increment (amount of underground part growth) over 2 weeks, fresh weight of plants, dry weight of plants, as shown in FIGS. 11, 12, 13, 14, 15, 16, in which the seedling height increment was 14.2cm, root length increment was 5.2cm, fresh weight of overground part plants was 3.057g, dry weight of overground part plants was 0.676g, fresh weight of underground part plants was 2.050g, dry weight of underground part plants was 0.278g, seedling height increment in over-expressed PePAE lines OE-1 and OE-2 was 114.08% (16.2 cm) and 111.03% (15.8 cm) of WT, root length increment was 109.62% (5.7 cm) and 112.82% (5.9 cm) of WT, fresh weight was 232.28% (7.100 g) and 274.05% (2 g) of overground part dry weight was 96% (96.676 g) and fresh weight of 35.35% (35.35.35 g) of dry weight of WT, respectively, and dry weight of underground part was 131.59% (35.35.35.35 g) of dry weight of the underground part was 35.35.35 g, respectively. It was demonstrated that over-expression PePAE6 increased the growth and biomass of OE-1 and OE-2 plants, i.e., expression PePAE6 promoted the growth of OE-1 and OE-2 plants.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (9)
1. The phyllostachys pubescens (Phyllostachys edulis) pectin acetyl esterase gene PePAE is characterized in that the nucleotide sequence of the phyllostachys pubescens (Phyllostachys edulis) pectin acetyl esterase gene is shown as SEQ ID NO. 1.
2. An isolated nucleic acid molecule, characterized in that it encodes a nucleotide sequence of the amino acid sequence shown in SEQ ID No. 2.
3. A biological material comprising the phyllostachys pubescens pectin acetylesterase gene PePAE of claim 1 or the isolated nucleic acid molecule of claim 2.
4. The biomaterial according to claim 3, wherein the biomaterial is a gene expression cassette, a gene expression vector, a gene cloning vector, an engineering bacterium or an engineering cell.
5. Use of the phyllostachys pubescens pectin acetylesterase gene PePAE of claim 1 or the isolated nucleic acid molecule of claim 2 or the biological material of claim 3 or 4 in one or more of the following applications: (1) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or an isolated nucleic acid molecule or biological material in reducing plant cell wall pectin acetylation level; (2) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or an isolated nucleic acid molecule or biological material in promoting pectin accumulation in plants; (3) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or an isolated nucleic acid molecule or biological material in promoting photosynthesis of plants; (4) The application of phyllostachys pubescens pectin acetylesterase gene PePAE or an isolated nucleic acid molecule or biological material in promoting plant growth.
6. The application according to claim 5, characterized in that the application (3) comprises one or more of the following applications: (a) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or an isolated nucleic acid molecule or biological material in improving net photosynthetic rate of plants; (b) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or an isolated nucleic acid molecule or biological material in improving the concentration of carbon dioxide between plant cells; (c) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or isolated nucleic acid molecules or biological materials in improving plant stomata conductivity; or the application (4) comprises one or more of the following applications: (a) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or isolated nucleic acid molecules or biological materials in increasing plant overground growth; (b) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or isolated nucleic acid molecules or biological materials in increasing plant underground growth; (c) Application of phyllostachys pubescens pectin acetyl esterase gene PePAE or an isolated nucleic acid molecule or biological material in increasing fresh weight of plants; (d) Use of phyllostachys pubescens pectin acetylesterase gene PePAE or an isolated nucleic acid molecule or biological material to increase plant dry weight.
7. Use of the phyllostachys pubescens pectin acetylesterase gene PePAE of claim 1 or the isolated nucleic acid molecule of claim 2 or the biological material of claim 3 or 4 for the cultivation of transgenic plants.
8. The use according to any one of claims 5 to 7, wherein the plant is phyllostachys pubescens or rice.
9. The method for extracting phyllostachys pubescens pectin acetylesterase gene PePAE as defined in claim 1, comprising the steps of: (1) Extracting RNA from phyllostachys pubescens leaves, and reversely transcribing the RNA into cDNA as a template; (2) And (3) performing PCR amplification by using a primer pair to obtain the phyllostachys pubescens pectin acetylesterase gene PePAE, wherein the upstream primer of the primer pair is shown as SEQ ID NO. 3, and the downstream primer of the primer pair is shown as SEQ ID NO. 4.
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