Phyllostachys edulis pectin acetyl esterase gene PePAE6 and extraction method and application thereof

Abstract

The present application belongs to the technical field of plant genetic engineering, and particularly relates to a moso bamboo pectin acetyl esterase gene PePAE6 as well as 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 pectin acetylation level of plant cell walls, promoting pectin accumulation in plants, improving the photosynthetic capacity of plants, and promoting the growth of plants. The moso bamboo pectin acetyl esterase gene PePAE6 is introduced into rice for verification, and the transgenic rice plants have a lower pectin acetylation level of cell walls, a higher pectin accumulation amount, a stronger photosynthetic capacity of leaves, and a faster and better growth than wild-type rice plants.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant genetic engineering, and particularly relates to a Phyllostachys edulis pectin acetyl esterase gene PePAE6 as well as an extraction method and application thereof. BACKGROUND

[0002] Pectin is a branched heteropolysaccharide rich in galacturonic acid, which mainly exists in the type I primary cell wall and intercellular layer of higher plants, and also exists in a small amount in the type II primary cell wall and secondary cell wall. Pectin is composed of four main components, namely homogalacturonan (HG), rhamnogalacturonan I (RGI), rhamnogalacturonan II (RGII), and xylogalacturonan (XGA). The four main components mainly participate in the composition of the matrix, so that the primary cell wall has elasticity and can be elongated with the elongation of the cell, and also participates in the remodeling of the cell wall and the expansion of the cell, further affecting or changing the individual development phenotype and physiological and biochemical processes of the plant.

[0003] The content of pectin in the primary cell wall of dicotyledonous plants accounts for about one-third of the dry weight; the content in herbaceous plants of monocotyledonous plants is relatively low, for example, in the family Poaceae. However, a large number of genes involved in pectin synthesis and modification still exist in the genome of monocotyledonous plants, which indicates that pectin also plays an important role in the individual growth and development process of monocotyledonous plants. Non-sugar groups are common groups for modifying pectin, such as methylesters (Me) and acetylesters (Ae), and the like. These groups enable pectin to be synthesized in a highly esterified state. And the various types of modified enzymes modified by these modification groups regulate the degree of esterification modification of plant components, change the physicochemical properties such as viscosity, hydrophobicity, and charge characteristics of plants, thereby regulating the overall structural properties of plant cell walls such as hardness, ductility, and porosity, and participating in the dynamic regulation activities of plant cell walls, so as to adapt to different growth and development stages of different tissues of themselves and respond to various stimuli and release of signal substances. Among them, acetylation modification of pectin by acetylesters is a common modification form of polysaccharide components in plant cell walls, and is also an important modification type of pectin.

[0004] As a plant pectin that can cleave acetyl ester bond to deacetylate pectin, pectin acetyl esterase (PAE; E.C. 3.1.1.6) determines the acetylation level of pectin and cell wall to a large extent. According to the CAZy database, PAE belongs to the CE13 family of carbohydrate esterases (CEs). After the first plant PAE was isolated from sweet orange (Citrus sinensis), researches have been widely carried out in Arabidopsis thaliana, Populus trichocarpa, Camellia sinensis and other species. For example, 12 PAE genes AtPAE members in Arabidopsis thaliana are divided into three branches, and three of the nine conserved motifs of AtPAEs (GCSxG, NxayDxwQ and HCQ) are speculated to be related to the catalytic mechanism. It is also found that the expression pattern of AtPAEs is consistent with that of pectin methyl esterase, pectin lyase and polygalacturonase related genes, but the specific function is unknown. Studies on reported mutants Pae8 and Pae9 in Arabidopsis thaliana show that PAE8 and PAE9 proteins play a role at the same time to remove one-third of the cell wall acetyl ester in the pectin formation process of Arabidopsis thaliana leaves, resulting in a significant reduction in the accumulation of cell wall acetyl ester, and further showing a decrease in flower stem height. At the same time, studies on PAE in Populus trichocarpa, Camellia sinensis, sweet orange and other plants show that plant PAE is highly conserved among different species, only has a single and typical PAE conserved domain. In addition, it is found in Populus trichocarpa that the expression of PAE is regulated by CO2 and nitrogen concentration levels. In sweet orange, CsPAE2 can improve the resistance of plants to bacterial wilt disease, indicating that plant PAE has functional diversity.

[0005] As one of the economically valuable grasses, bamboo has edible shoots and highly lignified bamboo culms. As a bamboo in bamboo, it is a representative of fast-growing plants, and the rapid growth of bamboo is closely related to the remodeling of cell wall and the dynamic optimization of cell wall polysaccharide properties. As a cell wall network structure matrix material, pectin is an indispensable component in the process of cell expansion and elongation, and is one of the basic conditions for the rapid growth of plants. At present, there is no related prior art on the mechanism of pectin acetylation and deacetylation of bamboo cell wall, and the influence of PAE on the whole cell wall and the resulting physiological and biochemical activities of plants is also blank. Therefore, identifying PAE in bamboo and revealing its molecular characteristics are of great significance to clarify the biological function of PAE in the growth and development of bamboo. SUMMARY

[0006] In order to solve the above problems, the application extracts the pectin acetyl esterase gene PePAE6 from the bamboo and finds that the gene has the functions of reducing the pectin acetylation level of the plant cell wall, promoting the pectin accumulation of the plant, improving the photosynthetic capacity of the plant and promoting the growth of the plant, and the gene can be used as a target gene to cultivate the plants with low pectin acetylation level of the cell wall, high pectin content, strong photosynthetic capacity and fast growth through the transgenic means.

[0007] In order to achieve the above purpose, the application can adopt the following technical scheme:

[0008] The application provides a bamboo (Phyllostachys edulis) pectin acetyl esterase gene PePAE6, and the nucleotide sequence of the bamboo pectin acetyl esterase gene PePAE6 is shown in SEQ ID NO: 1.

[0009] The application provides an isolated nucleic acid molecule, and the nucleotide sequence of the isolated nucleic acid molecule encodes the amino acid sequence shown in SEQ ID NO: 2.

[0010] The application provides a biological material, and the biological material comprises the bamboo pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule.

[0011] The application provides the bamboo pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or the biological material, and the bamboo pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or the biological material is applied to one or more of the following applications: (a) the bamboo pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or the biological material is applied to reducing the pectin acetylation level of the plant cell wall; (b) the bamboo pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or the biological material is applied to promoting the pectin accumulation of the plant; (c) the bamboo pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or the biological material is applied to promoting the photosynthesis of the plant; (d) the bamboo pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or the biological material is applied to promoting the growth of the plant.

[0012] The application provides the bamboo pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or the biological material, and the bamboo pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or the biological material is applied to cultivating the transgenic plants.

[0013] The application provides an extraction method of the bamboo pectin acetyl esterase gene PePAE6, and the extraction method comprises the following steps: (1) taking the bamboo leaves as the material, extracting the RNA and reversely transcribing the RNA into the cDNA as the template; (2) using a primer pair to perform PCR amplification to obtain the bamboo pectin acetyl esterase gene PePAE6, and the upstream primer of the primer pair is shown in SEQ ID NO: 3, and the downstream primer of the primer pair is shown in SEQ ID NO: 4.

[0014] The present application has at least the following beneficial effects: the pectin acetyl esterase gene PePAE6 of the present application has the functions of reducing the cell wall pectin acetylation level of the plant, promoting the pectin accumulation of the plant, improving the photosynthetic capacity of the plant, and promoting the growth of the plant; the pectin acetyl esterase gene PePAE6 of the bamboo is introduced into rice for verification, and the transgenic rice plants have lower cell wall pectin acetylation level, more pectin accumulation, stronger leaf photosynthetic capacity, and faster and better plant growth than the wild type rice plants. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is the agarose gel electrophoresis map of pCAMBIA1300-Ubi-PePAE6 recombinant plasmid double enzyme digestion in Example 2, wherein M1: DNA molecular weight marker DL15000; 1-3: expression plasmid;

[0016] Figure 2 It is the expression vector map of pCAMBIA1300-Ubi-PePAE6 in Example 2;

[0017] Figure 3 It is the PCR electrophoresis map of expression vector pCAMBIA1300-Ubi-PePAE6 plasmid transformed Agrobacterium monoclonal colony in Example 3, wherein M2: DNA molecular weight marker DL5000; 1: positive control with pCAMBIA1300-Ubi-PePAE6 plasmid as template; 2: negative control with pCAMBIA1300-Ubi empty vector plasmid as template; 3: negative control with water as template; 4-8: pCAMBIA1300-Ubi-PePAE6 transformed monoclonal colonies;

[0018] Figure 4 It is the PCR detection result electrophoresis map of PePAE6 in rice plants in Example 4, wherein M3: DNA molecular weight marker DL5000; 1-3: DNA extracted from PePAE6 overexpressed rice strain OE-1; 4-6: DNA extracted from PePAE6 overexpressed rice strain OE-2; 7-9: DNA extracted from wild type rice; 10: negative control with water as template; 11: negative control with pCAMBIA1300-Ubi empty vector plasmid as template; 12: positive control with pCAMBIA1300-Ubi-PePAE6 plasmid as template;

[0019] Figure 5 It is the representative picture of wild type rice and PePAE6 overexpressed rice strains OE-1 and OE-2 in Example 4;

[0020] Figure 6Figure 6 is a bar graph showing the comparison of cell wall pectin acetylation levels in PePAE6 transgenic rice of Example 5, with the amount of acetic acid released after saponification reaction in the extract, * indicates significant difference at the level of 0.01 < P < 0.05;

[0021] Figure 7 Figure 7 is a bar graph showing the comparison of pectin content in PePAE6 transgenic rice of Example 6, with the content of galacturonide, the main component of pectin in the extract, * indicates significant difference at the level of 0.01 < P < 0.05;

[0022] Figure 8 Figure 8 is a bar graph showing the comparison of net photosynthetic rate in PePAE6 transgenic rice of Example 7, * indicates significant difference at the level of 0.01 < P < 0.05;

[0023] Figure 9 Figure 9 is a bar graph showing the comparison of intercellular carbon dioxide concentration in PePAE6 transgenic rice of Example 7, * indicates significant difference at the level of 0.01 < P < 0.05, ** indicates significant difference at the level of P < 0.01;

[0024] Figure 10 Figure 10 is a bar graph showing the comparison of stomatal conductance in PePAE6 transgenic rice of Example 7, * indicates significant difference at the level of 0.01 < P < 0.05;

[0025] Figure 11 Figure 11 is a bar graph showing the comparison of plant height increment (aboveground growth) in PePAE6 transgenic rice of Example 8, ** indicates significant difference at the level of P < 0.01;

[0026] Figure 12 Figure 12 is a bar graph showing the comparison of root length increment (underground growth) in PePAE6 transgenic rice of Example 8, n.s. indicates P > 0.05;

[0027] Figure 13 Figure 13 is a bar graph showing the comparison of aboveground fresh weight of plant in PePAE6 transgenic rice of Example 8, ** indicates significant difference at the level of P < 0.01;

[0028] Figure 14 Figure 14 is a bar graph showing the comparison of aboveground dry weight of plant in PePAE6 transgenic rice of Example 8, ** indicates significant difference at the level of P < 0.01;

[0029] Figure 15 Figure 15 is a bar graph showing the comparison of underground fresh weight of plant in PePAE6 transgenic rice of Example 8, ** indicates significant difference at the level of P < 0.01;

[0030] Figure 16The bar chart is for analyzing the underground dry weight of the transgenic rice plants of PePAE6 in Example 8, and ** indicates significant difference at P≤0.01 level. DETAILED DESCRIPTION

[0031] The examples are provided to better illustrate the present application, but the present application is not limited to only the examples. Therefore, the skilled in the art can make non-essential improvements and adjustments to the embodiments according to the above description, which still belong to the protection scope of the present application.

[0032] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the disclosure. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, it is understood that the terms "comprises" and "comprising" are intended to indicate that there are features, numbers, operations, components, parts, elements, materials, or combinations thereof, present. The terms are disclosed in the specification, and are not intended to exclude the possibility of the presence or addition of one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof. As used herein, " / " can be interpreted as "and" or "or" depending on the situation.

[0033] The embodiment of the present application provides a Phyllostachys edulis pectin acetyl esterase gene PePAE6, and a nucleotide sequence of the Phyllostachys edulis pectin acetyl esterase gene PePAE6 is shown as SEQ ID NO: 1.

[0034] Another embodiment of the present application provides an isolated nucleic acid molecule, which encodes a nucleotide sequence of an amino acid sequence shown as SEQ ID NO: 2.

[0035] It should be noted that the nucleotide sequence capable of encoding the amino acid sequence as shown in SEQ ID NO: 2 is a sequence protected by the present application. For example, in addition to the sequence as shown in SEQ ID NO: 1, it can also be optimized from the sequence as shown in SEQ ID NO: 1. Specifically, the purpose of optimization is to improve the expression efficiency of the nucleotide sequence, and the optimization method is the existing technical means in the art, mainly including the following means: (1) such as the design and improvement of the expression vector for the transcription stage, such as the use of efficient expression elements such as promoters, enhancers, etc.; (2) improvement of the translation stage, optimization of the gene coding sequence of the recombinant protein itself, including codon bias optimization; (3) optimization of host cells, including efficient expression site positioning and targeted recombination; (4) optimization of cell large-scale culture process. Those skilled in the art can optimize the sequence as shown in SEQ ID NO: 1 according to specific needs. In some specific embodiments, the promoter or enhancer can be Ubipromoter or CaMV35Spromoter (enhanced); for codon optimization, briefly, due to the degeneracy of 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 codon" and "codon optimization" are developed. The specific optimization method can be referred to in "Presnyak, Vladimiret al. "Codon optimality is a major determinant of mRNA stability." Cell. 2015 Mar 12; 160(6): 1111-1124.".

[0036] Specifically, the sequence of SEQ ID NO: 1 is as follows:

[0037] ATGAAGACGAGGGAGAATGTTGGGATCACCAAATCCTGGGCTCATCTTCTTCTCCTTCTTGTGGTTGTTCTTGTCCTTGTGAGAAGCAGCGCGCAGGCGGCGGCCGACGAGCACAAGATCGGCGGCAGTAGGAGGCGGCGTGCGGCGGCGGCGGCGGCGCCGCCCGTTATGGTGCCTATCACCCTCCTCAGATCAGCCGTCGACAAGGGAGCTGTGTGCATGGATGGGACGCCGCCTGCTTACCACTTGGACCATGGCTCCGGGGCAGGGAACAGCAGCTGGATGATATTCCTAGAGGGAGGCGGGTGGTGCAACGACGTGTGGTCGTGCCGGTACCGTGCGGCGAGCCGGCTGGGCTCGTCGGATCGCATGGAGAAGCAGATCTACTTCGGGGGCATCAGGAGCGCCAACCCCCTCGACAACCCCGATTTCTACAACTGGAACCGGGTGATGATTCGCTACTGCGACGGCGCGTCCTTCGCCGGCGAAGGCTTCGACAAGGATCATGGGTTCTATTTCCGGGGCCAGCGCATCTGGGACGCGGTCGTCCGGCACCTCCTCTCCATCGGAATGGCCTCTGCAGATCAGGTGTTGCTCACCGGCGCCTCCGCCGGTGGACTGGCGGCCATCCTGCACTGCGACCAGTTCAGAGCCTTCTTCCCCGCCGCCACTGCCGGCGGCCGGAGCACCACCGTCAAGTGCCTCGCCGACGCAGGCCTCTTCCTCGACGCCGTGGATGTCTCCGGGGGCCGCAGCTTGAGATCGTACTACGGAGACGTCGTAGCCATGCAGGGGGTAGCTCAGAACCTGCCGCCGACTTGCACCGACCATCTGGACGCCACCTCGTGCTTCTTCCCTCAGAATATAATCGATGGCATAAACACCCCAATCTTCCTGCTAAATGCAGCATACGATGTCTGGCAGATCCAGCAAAGTTTGGCCCCAAACAAAGCTGACCCCAGCGGCGCCTGGCGAGCCTGCAAGTTCAACCGCTCAGCCTGCAATGCATCCCAGATGAAGTTCTTTCAAGAATTCAGGGGCCAGATGATAGCATCTGTGAAAGGTTTCTCCAGTTCCAAGAGCAACGGGTTGTTCATAAACTCGTGCTTCACTCACGGCCAGTCTGAGGCACCGGCCACCTGGAATAGTGCAGCTGGCTCTCCTGCTATTCAAAACAAGGGGATTGCAAAATCTGTTGGTGACTGGTACTTTGGTCGGGCTGAAGTGAAGGCGATCGACTGCCCTTATCCCTGCGACAAAACATGCCGTCACGATATA;

[0038] The sequence of SEQ ID NO: 2 is as follows:

[0039] MKTRENVGITKSWAHLLLLLVVVLVLVRSSAQAAADEHKIGGSRRRRAAAAAAPPVMVPITLLRSAVDKGAVCMDGTPPAYHLDHGSGAGNSSWMIFLEGGGWCNDVWSCRYRAASRLGSSDRMEKQIYFGGIRSANPLDNPDFYNWNRVMIRYCDGASFAGEGFDKDHGFYFRGQRIWDAVVRHLLSIGMASADQVLLTGASAGGLAAILHCDQFRAFFPAATAGGRSTTVKCLADAGLFLDAVDVSGGRSLRSYYGDVVAMQGVAQNLPPTCTDHLDATSCFFPQNIIDGINTPIFLLNAAYDVWQIQQSLAPNKADPSGAWRACKFNRSACNASQMKFFQEFRGQMIASVKGFSSSKSNGLFINSCFTHGQSEAPATWNSAAGSPAIQNKGIAKSVGDWYFGRAEVKAIDCPYPCDKTCRHDI.

[0040] It is to be noted that in some embodiments, the amino acid sequence as shown in SEQ ID NO: 2 includes a stop codon such as "TGA" at the end of the sequence as shown in SEQ ID NO: 1.

[0041] In another embodiment of the present application, a biological material is provided, which can include the Phyllostachys edulis pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule of the present application.

[0042] It is to be noted that the Phyllostachys edulis pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule of the present application can be prepared into products that are more suitable for practical application scenarios. For example, it can be prepared into a gene expression cassette, a gene expression vector, a gene cloning vector, an engineered bacterium or an engineered cell; the forms of the gene expression cassette, the gene expression vector, the gene cloning vector, the engineered bacterium or the engineered cell are known in the art. For example, the engineered bacterium can be Agrobacterium commonly used in plant cell genetic engineering, and the Phyllostachys edulis pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule of the present application is transduced into the Agrobacterium to prepare an engineered bacterium for expression of the gene PePAE6 or the nucleic acid molecule; for another example, the engineered cell is generally a tissue cell of a plant or a fertilized egg.

[0043] In another embodiment of the present application, the Phyllostachys edulis pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule of the present application or the biological material of the present application is used in one or more of the following applications: (a) the Phyllostachys edulis pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or the biological material is used to reduce the acetylation level of plant cell wall pectin; (b) the Phyllostachys edulis pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or the biological material is used to promote pectin accumulation in plants; (c) the Phyllostachys edulis pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or the biological material is used to promote photosynthesis in plants; (d) the Phyllostachys edulis pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or the biological material is used to promote plant growth.

[0044] In some embodiments, the application (3) can comprise one or more of the following applications: (a) application of the Phyllostachys edulis pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or biological material in increasing the net photosynthetic rate of the plant; (b) application of the Phyllostachys edulis pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or biological material in increasing the intercellular carbon dioxide concentration of the plant; (c) application of the Phyllostachys edulis pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or biological material in increasing the stomatal conductance of the plant; or the application (4) can comprise one or more of the following applications: (a) application of the Phyllostachys edulis pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or biological material in increasing the aboveground growth of the plant; (b) application of the Phyllostachys edulis pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or biological material in increasing the underground growth of the plant; (c) application of the Phyllostachys edulis pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or biological material in increasing the fresh weight of the plant; (d) application of the Phyllostachys edulis pectin acetyl esterase gene PePAE6 or the isolated nucleic acid molecule or biological material in increasing the dry weight of the plant.

[0045] It should be noted that the use of rice in the present application can be known that the content of pectin in the transgenic rice leaf transformed by the Phyllostachys edulis pectin acetyl esterase gene PePAE6 is 119.76% and 120.25% of the wild type rice respectively. It is shown that the pectin accumulation level of the overexpression PePAE6 strain OE-1 and OE-2 plants is higher than that of the wild type rice. It should be noted that the indicators for reducing the acetylation level of plant cell wall pectin, especially including the increase of plant acetic acid accumulation, that is, the product generated by the catalytic reaction in the process of PePAE6 reducing the acetylation level of plant cell wall pectin; the content of acetic acid released after the saponification reaction of the leaf extract of the transgenic rice transformed by the Phyllostachys edulis pectin acetyl esterase gene PePAE6 is 86.87% and 87.52% of WT respectively. It is shown that the acetylation level of cell wall pectin of the overexpression PePAE6 strain OE-1 and OE-2 plants is lower than that of WT; the net photosynthetic rate of the transgenic rice transformed by the Phyllostachys edulis pectin acetyl esterase gene PePAE6 is 115.92% and 118.05% of the wild type rice respectively, the intercellular carbon dioxide concentration is 102.84% and 104.02% of the wild type rice respectively, and the stomatal conductance is 138.31% and 143.04% of the wild type rice respectively. It is shown that the photosynthetic capacity of the transgenic rice transformed by the Phyllostachys edulis pectin acetyl esterase gene PePAE6 is promoted compared with the wild type rice; the aboveground growth of the transgenic rice transformed by the Phyllostachys edulis pectin acetyl esterase gene PePAE6 is 114.08% and 111.03% of the wild type rice respectively, the underground growth is 109.62% and 112.82% of the wild type rice respectively, the transgenic rice transformed by the Phyllostachys edulis pectin acetyl esterase gene PePAE6 increases the biomass at the same time, the aboveground fresh weight is 232.28% and 274.05% of the wild type rice respectively, the aboveground dry weight is 212.63% and 251.60% of the wild type rice respectively, the underground fresh weight is 262.28% and 300.33% of the wild type rice respectively, and the underground dry weight is 131.59% and 110.66% of the wild type rice respectively, which shows that the Phyllostachys edulis pectin acetyl esterase gene PePAE6 can promote plant growth. It should be noted that the aspects of promoting plant growth, especially including increasing plant growth and increasing plant biomass.

[0046] It should also be noted that based on the Phyllostachys edulis pectin acetyl esterase gene PePAE6 having the above-mentioned functions of reducing the acetylation level of plant cell wall pectin, increasing pectin accumulation, enhancing leaf photosynthetic capacity and promoting plant growth, the optimized separable nucleic acid molecule and the biological material loaded with the gene PePAE6 and the separable nucleic acid molecule also have the above-mentioned functions.

[0047] Another embodiment of the present application provides an application of the Phyllostachys edulis pectin acetyl esterase gene PePAE6 in the present application or the isolated nucleic acid molecule in the present application or the biological material in the present application in cultivating transgenic plants.

[0048] It should be noted that, as described above, the Phyllostachys edulis pectin acetyl esterase gene PePAE6 in the present application or the isolated nucleic acid molecule in the present application or the biological material in the present application has the functions of reducing the cell wall pectin acetylation level of the plant, increasing the pectin accumulation amount, enhancing the photosynthetic capacity of the leaf, and promoting the growth of the plant; the Phyllostachys edulis pectin acetyl esterase gene PePAE6, the nucleic acid molecule or the biological material can be transferred into the plant by transgenic technology (including gene editing technology) for cultivation, so that the plant with low cell wall pectin acetylation level, the high-pectin biomass plant, the high-photosynthetic capacity plant and the high-growth plant can be obtained.

[0049] Specifically, the terms “plant with low cell wall pectin acetylation level”, “high-pectin biomass plant”, “high-photosynthetic capacity plant” and “high-growth plant” refer to that the transgenic plant obtained by transferring the Phyllostachys edulis pectin acetyl esterase gene PePAE6 in the present application as a target gene is better than the wild-type plant under the same conditions in the corresponding indicators (including galacturonic acid content, acetic acid release amount, net photosynthetic rate, intercellular carbon dioxide concentration, stomatal conductance, increment of seedling height, increment of root length, fresh weight and dry weight of aboveground part, fresh weight and dry weight of underground part) detected under the same detection method.

[0050] In some specific embodiments, the plant in the above application can be Phyllostachys edulis or rice. It should be noted that the plant described above can be a plant known in the art, such as Phyllostachys edulis or rice.

[0051] It should be understood that after the Phyllostachys edulis pectin acetyl esterase gene PePAE6 is optimized to improve the expression efficiency according to the above method, the Phyllostachys edulis obtained by constructing a vector and transferring it into the Phyllostachys edulis, artificially increasing or decreasing the transcription level of the PePAE6 gene, and then cultivating can be a transgenic Phyllostachys edulis.

[0052] Another embodiment of the present application provides an extraction method of the Phyllostachys edulis pectin acetyl esterase gene PePAE6 in the present application, which can include: (1) taking Phyllostachys edulis leaves as the material, extracting RNA, and reverse transcribing into cDNA as a template; (2) using a primer pair to perform PCR amplification to obtain the Phyllostachys edulis pectin acetyl esterase gene PePAE6, 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.

[0053] It should be noted that the extraction method of the bamboo pectin acetylesterase gene PePAE6 in this invention can be carried out using methods known in the art, such as PCR amplification extraction. The primers for PCR amplification extraction can be designed according to conventional design methods in the art. Preferably, the upstream primer is shown in SEQ ID NO:3 and the downstream primer is shown in SEQ ID NO:4. Under the amplification of this primer pair, a large amount of bamboo pectin acetylesterase gene PePAE6 can be obtained.

[0054] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0055] Example 1: Obtaining the coding region sequence of the bamboo pectin acetylesterase gene PePAE6

[0056] RNA was extracted from bamboo shoots (Phyllostachys edulis) and reverse transcribed into cDNA as a template. Specifically, the RNA was first reverse transcribed into cDNA using a kit (Jianshi Biosciences, TR205-D, Beijing), and then the steps of the PrimeScript RT Reverse Transcription Kit (Takara, RR037A, Japan) were followed.

[0057] Specific primers were designed based on the predicted gene PH02Gene08079 in moso bamboo, and the coding region sequence was amplified by PCR. The primer sequences are shown in Table 1 below. The amplification system and amplification program were based on the instructions of 2×Taq Master Mix (Dye Plus) (Novazia, P112-01, Nanjing).

[0058] Table 1 PCR amplification primers

[0059] Upstream primer 5'-GCTGATATGAAGACGAGGGAGAATG-3' (SEQ ID NO: 3) Downstream primer 5'-CTTCTCATCATATGTCGTGACGGC-3' (SEQ ID NO: 4)

[0060] The PCR amplification products were detected by agarose gel electrophoresis. The target band was excised, purified, and recovered. The recovered DNA fragment was ligated into the pGEM-TEasy vector and transformed into *E. coli* DH5α competent cells. After blue-white screening, positive clone plasmids were extracted and analyzed for restriction enzyme digestion. Single clones were then sequenced, revealing an insert fragment of 1281 bp, as shown in SEQ ID NO:1. Subsequently, the Blast online software was used to compare the sequence with tens of thousands of genes predicted in the *Phyllostachys edulis* genome. The sequence similarity of SEQ ID NO:1 to PH02Gene08079 was found to be the highest, at 95.44%.

[0061] Further analysis by BlastP online comparison found that the amino acid sequence (SEQ ID NO: 2) encoded by the gene had the highest consistency with PAE (XM_039958098.1) of Panicum virgatum (83.23%), with PAE (XM_025961332.1) of Panicum hallii (82.42%), and with PAE (XM_045111292.1) of Hordeum vulgare (81.85%); protein domain analysis showed that the protein had a PAE enzyme-related conserved domain PAE (pfam03283). It can be seen that the cloned gene encodes a pectin acetyl esterase, and the gene is named PePAE6, and the pGEM-T Easy vector containing the gene is pT-PePAE6.

[0062] Example 2 Construction of a plant expression vector carrying the Phyllostachys edulis pectin acetyl esterase gene PePAE6

[0063] According to the sequence shown in SEQ ID NO: 1, an amplification primer with a homologous arm and a restriction site BamH I / Pml I was designed, and the obtained plasmid of Example 1 was diluted 40 times as a template. PrimeSTAR Max High-Fidelity Enzyme was used for PCR amplification of the fragment containing the restriction site (BamH I / Pml I) and the deoxyribonucleotide sequence of the coding region of Phyllostachys edulis PePAE6 required for expression vector construction; the primer sequences are shown in Table 2; the amplification system and the amplification program refer to the instructions of Max DNA Polymerase (Takara, R045). Max DNA Polymerase (Takara, R045).

[0064] Table 2 Primer sequences

[0065]

[0066] The PCR amplification product was detected by agarose gel electrophoresis, and the target band was purified and recovered. The target gene and the pCAMBI1300-Ubi vector restriction enzyme fragments were connected using one-step cloning technology, and the reaction system (20 μL) was as follows.

[0067] Table 3 Cloning reaction system of target gene and pCAMBI1300-Ubi vector restriction enzyme fragments

[0068] Component Volume 5x reaction buffer 4.0 μL NovoRec Plus recombinase 1.0 μL pCAMBI 1300-Ubi restriction vector 1.0 μL Fragment of interest 1.0 μL ddH2O 13.0 μL

[0069] All reagents were added to the PCR tube in the above order, mixed and centrifuged, placed in a 50°C water bath for 10 min, and then transformed into E. coli DH5α. The plasmid was extracted, and the restriction map identification and sequencing verification were carried out. The restriction map identification is as follows:Figure 1 The obtained recombinant expression vector was named as pCAMBIA1300-Ubi-PePAE6 (see Figure 2 ).

[0070] Example 3 Identification of monoclonal colonies containing expression vector pCAMBIA1300-Ubi-PePAE6

[0071] The expression vector pCAMBIA1300-Ubi-PePAE6 constructed in Example 2 was transformed into competent cells of Agrobacterium tumefaciens strain GV3101 by freeze-thaw method, and single colonies grown on kanamycin-resistant (50 mg·L -1 ) plates were picked for PCR identification. The monoclonal colonies formed after transformation with the PePAE6 gene recombinant expression vector were used as templates for PCR detection with the primers in Example 2 (shown in Table 2), and the recombinant plasmid pCAMBIA1300-Ubi-PePAE6 was used as a positive control, and pCAMBIA1300-Ubi empty vector and water were used as negative controls.

[0072] The results are shown in Figure 3 , and the PCR electrophoresis results of the Agrobacterium monoclonal colonies transformed with the expression vector pCAMBIA1300-Ubi-PePAE6 plasmid showed that the monoclonal colonies contained the target gene fragment, and the Agrobacterium monoclonal bacterial solution obtained by shaking could be used for infection transformation experiments.

[0073] Example 4 Transformation of PePAE6 into rice and PCR detection

[0074] Using the bacterial solution obtained in Example 3, wild-type rice (WT) (Nipponbare, Oryza sativa L. spp. japonica) callus was infected, and through continuous selection for resistance (hygromycin 50 mg·L -1 ), two transgenic rice lines were finally obtained, and gene expression detection was performed.

[0075] The DNA of the transgenic rice plants and wild-type rice plants was extracted, and PCR detection was performed using the primers in Example 2. The results are shown in Figure 4 , which showed that the target gene was detected in the transgenic rice plants, but not in the wild-type rice plants, indicating that PePAE6 had been transformed into the rice plants. After 3 weeks of culture (hydroponics) under suitable conditions (28℃±2℃ in a light incubator, light for 12h, dark for 12h, relative humidity 60%, in Kikuchi B rice nutrient solution (Coolab, NS1050, Beijing)), rice seedlings that could be used for subsequent experiments were obtained, as shown in Figure 5 .

[0076] Example 5 Analysis of the level of cell wall pectin acetylation in PePAE6 transgenic rice

[0077] The content of product acetate after saponification reaction of pectin extract in rice leaves in Example 4 was determined, and the results are shown in Table 2. Figure 6 As shown in Table 2, the content of product in WT was 5.208 g / 100 g, and the content of product in overexpression PePAE6 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 shown that overexpression of PePAE6 reduced the level of cell wall pectin acetylation in OE-1 and OE-2 plants.

[0078] Example 6 Analysis of pectin content in PePAE6 transgenic rice

[0079] The content of pectin in rice leaves in Example 4 was determined, and the results are shown in Table 3. Figure 7 As shown in Table 3, the content of galacturonic acid in WT was 3.311 μmol / g, and the content of galacturonic acid in overexpression PePAE6 lines OE-1 and OE-2 was 119.76% (3.965 μmol / g) and 120.25% (3.982 μmol / g) of WT, respectively. It was shown that overexpression of PePAE6 improved pectin accumulation in OE-1 and OE-2 plants.

[0080] Example 7 Comparative analysis of photosynthetic physiological parameters in PePAE6 transgenic rice

[0081] After detecting the photosynthetic parameters of 3-week-old rice seedlings using a portable photosynthesis instrument and statistical analysis, it was found that, as shown in Table 4, Figure 8 , Figure 9 , Figure 10 the net photosynthetic rate in WT was 14.663 μmol m - 2s - 1, the intercellular carbon dioxide concentration was 278.278 μmol mol - 1, and the stomatal conductance was 0.234 mol m - 2s - 1, the net photosynthetic rate in overexpression PePAE6 lines OE-1 and OE-2 was 115.92% (16.998 μmol m - 2s - 1) and 118.05% (17.310 μmol m - 2s - 1) of WT, respectively, and the intercellular carbon dioxide concentration was 102.84% (286.171 μmol mol -1) and 104.02% (289.468 μmol mol - 1), respectively, which were 138.31% (0.324 mol m - 2s - 1) and 143.04% (0.335 mol m - 2s - 1), respectively. It was shown that overexpression of PePAE6 improved the photosynthetic capacity of OE-1 and OE-2 plants.

[0082] Example 8: Comparison of phenotype and biomass of PePAE6 transgenic rice

[0083] The physiological phenotype of 3-week-old rice seedlings was statistically analyzed, including the increment of seedling height (aboveground growth) in 2 weeks, the increment of root length (underground growth) in 2 weeks, fresh weight of plants, and dry weight of plants, as shown in Table 1. Figure 11 、 Figure 12 、 Figure 13 、 Figure 14 、 Figure 15 、 Figure 16 The increment of seedling height in WT was 14.2 cm, the increment of root length was 5.2 cm, the fresh weight of aboveground plants was 3.057 g, the dry weight of aboveground plants was 0.676 g, the fresh weight of underground plants was 2.050 g, and the dry weight of underground plants was 0.278 g. The increment of seedling height in OE-1 and OE-2 was 114.08% (16.2 cm) and 111.03% (15.8 cm) of WT, respectively, the increment of root length was 109.62% (5.7 cm) and 112.82% (5.9 cm) of WT, respectively, the fresh weight of aboveground plants was 232.28% (7.100 g) and 274.05% (8.377 g) of WT, respectively, the dry weight of aboveground plants was 212.63% (1.437 g) and 251.60% (1.700 g) of WT, respectively, the fresh weight of underground plants was 262.28% (5.377 g) and 300.33% (6.157 g) of WT, respectively, and the dry weight of underground plants was 131.59% (0.663 g) and 110.66% (0.730 g) of WT, respectively. It was shown that overexpression of PePAE6 improved the growth and biomass of OE-1 and OE-2 plants, i.e., expression of PePAE6 promoted the growth of OE-1 and OE-2 plants.

[0084] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the spirit and scope of the present application, and all of them should be covered in the scope of the claims of the present application.

Claims

1. The pectin acetylesterase gene PePAE6 from moso bamboo (Phyllostachys edulis), characterized by, Its nucleotide sequence is shown in SEQ ID NO:

1.

2. An isolated nucleic acid molecule, characterized in that, Its nucleotide sequence encodes the amino acid sequence shown in SEQ ID NO:

2.

3. A biomaterial, characterized in that, It includes the bamboo pectin acetylesterase gene PePAE6 as described in claim 1 or the isolated nucleic acid molecule as described in claim 2; the biological material is a gene expression cassette, a gene expression vector, or engineered bacteria.

4. Overexpression of the bamboo pectin acetylesterase gene PePAE6 as described in claim 1 or the isolated nucleic acid molecule as described in claim 2 in one or more of the following applications: (1) application in reducing the level of pectin acetylation in rice cell walls; (2) application in promoting pectin accumulation in rice; (3) application in promoting photosynthesis in rice; (4) application in promoting rice growth; application (4) includes one or more of the following applications: (a) application in increasing the aboveground growth of rice; (b) application in increasing the underground growth of rice; (c) application in increasing the fresh weight of rice; (d) application in increasing the dry weight of rice.

5. The application according to claim 4, characterized in that, Application (3) includes one or more of the following applications: (a) application in increasing the net photosynthetic rate of rice; (b) application in increasing the intercellular carbon dioxide concentration of rice; (c) application in increasing the stomatal conductance of rice.

6. The method for extracting the bamboo pectin acetylesterase gene PePAE6 according to claim 1, characterized in that, include: (1) RNA was extracted from bamboo leaves and reverse transcribed into cDNA as a template; (2) The gene PePAE6 of bamboo pectin acetylesterase was obtained by PCR amplification using primer pair. The upstream primer of the primer pair is shown in SEQ ID NO:3, and the downstream primer of the primer pair is shown in SEQ ID NO:4.

Images